pcr experiment result

Understanding your PCR nasal swab test results

Update: The current turnaround time is averaging 2-3 days to receive your COVID-19 PCR (nasal swab) results.

All PCR testing is performed by one of our commercial lab partners. The testing platforms used are Roche Cobus or Hologic Panther, both with Emergency Use Authorization by the FDA. Both platforms search for 2 targets; target 1 that is specific to SARS-CoV-2 (the coronavirus causing COVID-19) and target 2 for general Corona Viruses (including but not limited to SARS-CoV-2).

Positive results: You have tested positive for Sars-CoV-2, the virus causing COVID-19. Either target 1 alone or both targets 1 and 2 were detected (our lab partners do not specifically call out if you tested positive for target 1 alone or target 1 and 2 as it is not relevant, either scenario is positive). You are likely actively contagious and should home quarantine (sleep alone in bed, if possible use your own bathroom, wipe down surfaces, and wear a mask when in the same room as others). Please note, a small percentage of patients with active infection may be completely asymptomatic. CityMD recommends the CDC's most up to date return to work recommendation of the rule of 10/3. If you did not have symptoms at the time of your PCR nasal swab, you may return to work in 10 days (provided you do not have a fever 3 days prior to return to work). If you are asymptomatic and test positive, you are still likely contagious to others. A positive PCR test does not yield any information about potential immunity. Please be re-evaluated immediately for worsening symptoms such as shortness of breath or lightheadedness.

Negative results: With a high likelihood, the results state you were not infected with Sars-CoV-2 at the time of testing. Neither target 1 or target 2 were detected. We recommend following quarantine recommendations and universal precautions (hand washing, social distancing, and when appropriate PPE such as masks and gloves). This test does not give information about past infections or future immunity.

Inconclusive results (presumptive positive) or presumed positive: Inconclusive/Presumptive Positive or Presumed Positive means target 1 was not detected but target 2 was detected. It is presumed if you had symptoms consistent with COVID-19 and test positive for target 2, you have COVID-19. You are likely actively contagious and should home quarantine (sleep alone in bed, if possible use your own bathroom, wipe down surfaces, and wear a mask when in the same room as others). Please note, a small percentage of patients with active infection may be completely asymptomatic. CityMD recommends the CDC's most up to date return to work recommendation of the rule of 10/3. If you did not have symptoms at the time of your PCR nasal swab, you may return to work in 10 days (provided you do not have a fever 3 days prior to return to work). A positive PCR test does not yield any information about potential immunity. Please be re-evaluated immediately for worsening symptoms such as shortness of breath or lightheadedness.

Sunrise Labs will report your test as:

Quest Labs will report your test as:

If you have any questions or need to discuss your results further, please call Aftercare at 844.824.8963 Extension 8120. We apologize as we are currently experiencing high call volumes to our Aftercare Department. We appreciate your patience and understanding during this time.

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Polymerase Chain Reaction (PCR) : Principle, Procedure, Components, Types and Applications

The polymerase chain reaction (PCR) is a laboratory technique for DNA replication that allows a “target” DNA sequence to be selectively amplified. PCR can use the smallest sample of the DNA to be cloned and amplify it to millions of copies in just a few hours.

Discovered in 1985 by Kerry Mullis, PCR has become both and essential and routine tool in most biological laboratories.

Principle of PCR

The PCR involves the primer mediated enzymatic amplification of DNA. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand.

Primer is needed because DNA polymerase can add a nucleotide only onto a preexisting 3′-OH group to add the first nucleotide. DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extended region of double stranded DNA.

Components of PCR

The PCR reaction requires the following components:

DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.
DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70°C.
Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.
Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.
Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity.

Procedure of PCR

All the PCR components are mixed together and are taken through series of 3 major cyclic reactions conducted in an automated, self-contained thermocycler machine.

Denaturation : This step involves heating the reaction mixture to 94°C for 15-30 seconds. During this, the double stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds.
Annealing : The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.
Elongation : Also known at extension, this step usually occurs at 72-80°C (most commonly 72°C). In this step, the polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′ direction. Under optimal conditions, DNA polymerase will add about 1,000 bp/minute.

With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA.

These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Types of PCR

In addition to the amplification of a target DNA sequence by the typical PCR procedures already described, several specialised types of PCR have been developed for specific applications.

Real-time PCR
Quantitative real time PCR (Q-RT PCR)
Reverse Transcriptase PCR (RT-PCR)
Multiplex PCR
Long-range PCR
Single-cell PCR
Fast-cycling PCR
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR
In situ PCR
Variable Number of Tandem Repeats (VNTR) PCR
Asymmetric PCR
Repetitive sequence-based PCR
Overlap extension PCR
Assemble PCR
Intersequence-specific PCR(ISSR)
Ligation-mediated PCR
Methylation –specifin PCR
Miniprimer PCR
Solid phase PCR
Touch down PCR, etc

Applications of PCR

Some common applications of PCR in various fields can be explained in following categories.

Medical Applications:

Genetic testing for presence of genetic disease mutations. Eg: hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism
Detection of disease causing genes in suspected parents who act as carriers.
Study of alteration to oncogenes may help in customization of therapy
Can also be used as part of a sensitive test for tissue typing, vital to organ transplantation
Helps to monitor the gene in gene therapy

Infectious disease Applications:

Analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, tuberulosis etc.
Detection of new virulent subtypes of organism that is responsible for epidemics.

Forensic Applications:

Can be used as a tool in genetic fingerprinting. This technology can identify any one person from millions of others in case of : crime scence, rule out suspects during police investigation, paternity testing even in case of avaibility of very small amount of specimens ( stains of blood, semen, hair etc)

Research and Molecular Genetics:

In genomic studies: PCR helps to compare the genomes of two organisms and identify the difference between them.
In phylogenetic analysis. Minute quantities of DNA from any source such a fossilized material, hair, bones, mummified tissues.
In study of gene expression analysis, PCR based mutagenesis
In Human genome project for aim to complete mapping and understanding of all genes of human beings.

Frequently Asked Questions

Q 1. what is the purpose of a polymerase chain reaction.

It is a quick and inexpensive method of amplifying small segments of DNA, which is essential for molecular and genetic analyses. Every study of isolated DNA pieces needs to undergo polymerase chain reaction amplification.

Q 2. What happens in a polymerase chain reaction?

A segment of DNA is amplified using PCR. To do so, the sample is heated to denature the DNA. By denaturing means separating DNA segments into two pieces of single-stranded DNA. The enzyme Taq polymerase synthesizes the DNA to build to new strands resulting in the duplication of two original DNA. each of the strands is used to create two new copies – the cycle can be repeated 40 times making it possible to build a billion copy of the original DNA segment. The entire process would only take a few hours to complete.

Q 3. What is needed for PCR?

To be able to perform PCR, the following is needed: 1. DNA sample 2. ddNTPs (free nucleotides) 3. DNA primers 4. DNA polymerase

Q 4. How is the PCR used to diagnose?

– It is used to count the number of DNA/copies of a gene present in a given sample. – It is used to find out the viral load of HIV in patients suffering from AIDS. – It is helpful in determining the number of cancerous cells that are remaining in a cancer patient undergoing treatment.

Q 5. Is real-time PCR quantitative?

Real-time PCR is also called quantitative PCR. It is based on the method that includes amplification of the target DNA sequence and quantifying the concentration of DNA species in the reaction.

Q 6. What is the end result of PCR?

The end product of the polymerase chain reaction is a brand new DNA strand with a double-stranded DNA molecule.

Q 7. What happens at 72 degrees in PCR?

The 72 degrees’ temperature is the optimum for Taq polymerase. Once it reaches this temperature, the extension process begins. Taq polymerase works off the primers and will generate a new strand of DNA which results in double-stranded DNA.

Q 8. How many types of PCR are there?

Not all PCRs are the same. You will be surprised to know that there are many types of PCR and the most common ones are the following: Real-time PCR/Quantitative PCR/qPCR – It uses a fluorescent dye to tag the molecules of DNA. it is used to detect and quantify PCR products in real-time. Multiplex PCR – It multiplies multiple fragments in a single sample of DNA using a number of primers. reverse-transcriptase – The purpose is to create complementary DNA by means of reverse transcribing RNA to DNA with the help of reverse transcriptase. Hot start PCR – Heat is used to denature antibodies that are used for Taq polymerase inactivation. Nested PCR – Once the initial PCR cycle is done, another PCR is done but this time with the use of a new primer nested within the original primer. Thus, the term nested PCR. The reason for doing so is to reduce the risk of unwanted products. Assembly PCR – Overlapping primers are used to amplify longer fragments of DNA. Long-range PCR – A longer range of DNA is formed with the help of a polymerase mixture. In situ PCR – It is a type of PCR that takes place in the cells or fixed tissue on a slide. Asymmetric PCR – A single stand of target DNA is amplified.

Q 9. How accurate is a polymerase chain reaction?

The polymerase chain reaction is a highly sensitive procedure. the sensitivities range from 61% to 100%. The specificities range from 11% to 100%.

Q 10. What diseases can PCR detect?

There are different types of diseases that can be detected using PCR such as: 1. Hepatitis 2. HIV 3. Human papillomavirus (causes genital warts and cervical cancer) 4. Malaria 5. Anthrax 6. Epstein-Barr virus in people with glandular fever

Q 11. What do PCR primers do?

They are short fragments of single-stranded DNA, around 15 to 30 nucleotides long complementary to sequences of DNA that flank to the target region. What does a PCR primer do? It provides a free 3’ –OH group where DNA polymerase can easily add dNTPs.

Q 12. Why is PCR important?

A polymerase chain reaction is important as once DNA is amplified it can be used in various laboratory procedures and clinical methods. Examples are fingerprinting of DNA, diagnosis of various genetic disorders, detecting the presence of bacteria and viruses such as in the case of people with HIV/AIDS.

Q 13. What enzyme is used for PCR?

An enzyme is used to complete the polymerase chain reaction. The two enzymes used are DNA polymerase enzyme and Taq enzyme. The DNA polymerase enzyme is used to create new strands of DNA with the use of existing strands as templates.

Q 14. What are the 4 steps of PCR?

The polymerase chain reaction is composed of four primary steps: 1. The first step is denaturation using heat. 2. The second step is annealing the primer to a specific target sequence of DNA. Extension 3. End of the first cycle.

Q 15. Who first got the idea of a polymerase chain reaction?

The polymerase chain reaction is a product of the inventive mind of Kary B. Mullis. He invented this procedure in 1985 which paved a way to scientists making millions of copies of scarce DNA samples.

Q 16. Why it is called real-time PCR?

It is called real-time PCR primarily because it monitors the progress of polymerase chain reaction in real-time. only a small amount of PCR product can be quantified during the procedure.

Q 17. Is the RT PCR expensive?

The RT PCR test is an expensive procedure. it is a nuclear-derivative way of identifying the presence of specific genetic materials from a particular pathogen such as the virus. Why it is expensive? It is primarily because the equipment and resources used to run the test are scarce.

Q 18. What is the difference between real-time PCR and PCR?

The difference between traditional PCR and real-time PCR is that the former has advanced from detection at the end-point of the reaction to detection. On the other hand, the latter enables the detection of PCR amplification during the early stage of the polymerase chain reaction.

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Testing for COVID-19

What to know.

COVID-19 testing can help you know if you have COVID-19 so you can decide what to do next, like getting treatment to reduce your risk of severe illness and taking steps to lower your chances of spreading the virus to others.

Types of tests

Viral tests look for a current infection with SARS-CoV-2, the virus that causes COVID-19, by testing specimens from your nose or mouth.

There are two main types of viral tests.

Nucleic Acid Amplification Tests (NAATs)

Healthcare professional administering a NAAT test.

Nucleic acid amplification tests (NAATs), including PCR tests, are more likely to detect the virus than antigen tests. NAATs tests are the “gold standard” for COVID-19 tests.

Your sample will usually be taken by a healthcare provider and transported to a laboratory for testing and may take up to 3 days to receive results. Some NAATs may be performed at the point-of-care and provide results more quickly.

Antigen Tests

Antigen tests* are rapid tests that usually produce results in 15-30 minutes. Positive results are accurate and reliable. However, in general, antigen tests are less likely to detect the virus than NAAT tests, especially when symptoms are not present. Therefore, a single negative antigen test cannot rule out infection.

To be confident you do not have COVID-19, FDA recommends 2 negative antigen tests for individuals with symptoms or 3 antigen tests for those without symptoms, performed 48 hours apart. A single NAAT test can be used to confirm an antigen test result.

*Self-tests, or at-home tests, are antigen tests that can be taken anywhere without having to go to a specific testing site. Read self-test package inserts thoroughly and follow the instructions closely when performing the test.

When you get tested

Choose the right type of test for your circumstance.
When using an antigen test, follow the recommendations provided by FDA and the test's manufacturer.
If you do not, your results may be less likely to correctly indicate whether you have COVID-19 or not.

Choosing a COVID-19 test

I want to get tested and:, i have not had covid-19 or i have not had a positive test within the past 90 days..

You may choose a NAAT, including PCR, or antigen test. If you use an antigen test and your result is negative, repeat testing following FDA recommendations .

I tested positive for COVID-19 in the last 90 days.

My first positive test result was within 30 days or less:.

I have symptoms: Use an antigen test. Repeat negative tests following FDA recommendations .
I do not have symptoms: Testing is not recommended to detect a new infection.

My first positive test result was within 31-90 days:

I do not have symptoms: Use an antigen test. Repeat negative tests following FDA recommendations .

After a positive test result, you may continue to test positive for some time. Some tests, especially NAAT tests, may continue to show a positive result for up to 90 days.

Reinfections can occur within 90 days, which can make it hard to know if a positive test indicates a new infection.

Consider consulting a healthcare provider if you have any questions or concerns about your circumstances.

Getting a COVID-19 test

Buy self-tests (at-home tests).

Buy self-tests (at-home tests) online or in pharmacies and retail stores. If you have health insurance, it may reimburse the cost of purchasing self-tests. Visit FDA's website for a list of authorized tests.

Go to a testing location

Visit a community-based testing location , such as a pharmacy or health center near you. These locations may offer NAAT, including PCR, or antigen tests, and provide low- or no-cost testing for everyone, including people without insurance. Free NAAT or antigen tests may also be available through your local health department .
Talk to a doctor or healthcare provider about other testing options that may be available to you.
If you are a person with a disability, the Disability Information and Access Line can help you access a test or find a test location.

Order Your 4 Free At-Home COVID-⁠19 Tests‎‎

Interpreting your results, if your covid-19 test is positive.

A positive COVID-19 test means the virus was detected and you have or recently had an infection .

Take steps to prevent spreading COVID-19.
Monitor your symptoms . If you have any emergency warning signs , seek emergency care immediately.
Seek health care right away for treatment if you have risk factors for severe illness. Treatment may be an option to make your symptoms less severe and shorten the time you are sick. Treatment needs to be started within a few days of when your symptoms begin.

If your COVID-19 test is negative

A negative COVID-19 test means the test did not detect the virus, but this doesn't rule out that you could have an infection . If you used an antigen test, follow FDA recommendations for repeat testing .

You may have COVID-19 but tested before the virus was detectable.
You may have another viral infection or illness.
Take actions to help protect yourself and others from health risks caused by respiratory viruses.
Contact a healthcare provider if you have any questions about your test result.

COVID-19 (coronavirus disease 2019) is a disease caused by a virus named SARS-CoV-2. It can be very contagious and spreads quickly.

For Everyone

Health care providers, public health.

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Understanding COVID-19 PCR Testing

Beginning with the Human Genome Project 30 years ago, NHGRI has supported research that reduced the cost and increased the speed of genetic and genomic sequencing, enabling the rapid pivot towards COVID-19 research and development. NHGRI’s investments in DNA-sequencing and related technologies created a foundation that allowed companies to rapidly deploy COVID-19 PCR diagnostic testing early in the pandemic.

Key Points:

Genomic research has been central to understanding and combating the SARS-CoV-2 (COVID-19) pandemic.
Polymerase chain reaction (PCR) is a laboratory technique that uses selective primers to “copy” specific segments of a DNA sequence.
COVID-19 PCR tests use primers that match a segment of the virus’s genetic material. This allows many copies of that material to be made, which can be used to detect whether or not the virus is present.
A positive COVID-19 PCR test means that SARS-CoV-2 is present. A negative result could either mean that the sample did not contain any virus or that there is too little viral genetic material in the sample to be detected.

What is PCR?

Polymerase chain reaction (PCR) is a common laboratory technique used in research and clinical practices to amplify, or copy, small segments of genetic material. PCR is sometimes called “molecular photocopying,” and it is incredibly accurate and sensitive. Short sequences called primers are used to selectively amplify a specific DNA sequence. PCR was invented in the 1980s and is now used in a variety of ways, including DNA fingerprinting, diagnosing genetic disorders and detecting bacteria or viruses. Because molecular and genetic analyses require significant amounts of a DNA sample, it is nearly impossible for researchers to study isolated pieces of genetic material without PCR amplification.

How does COVID-19 PCR testing work?

COVID-19 testing uses a modified version of PCR called quantitative polymerase chain reaction (qPCR). This method adds fluorescent dyes to the PCR process to measure the amount of genetic material in a sample. In this instance, healthcare workers measure the amount of genetic material from SARS-CoV-2.

The testing process begins when healthcare workers collect samples using a nasal swab or saliva tube. The SARS-CoV-2 virus, which is the pathogen that causes COVID-19, uses RNA as its genetic material. First, the PCR is converted from single-stranded RNA to double-stranded DNA in a process called reverse transcription. The two DNA template strands are then separated.

Primers attach to the end of these strands. Primers are small pieces of DNA designed to only connect to a genetic sequence that is specific to the viral DNA, ensuring only viral DNA can be duplicated (right). After the primers attach, new complementary strands of DNA extend along the template strand. As this occurs, fluorescent dyes attach to the DNA, providing a marker of successful duplication. At the end of the process, two identical copies of viral DNA are created. The cycle is then repeated 20-30 times to create hundreds of DNA copies corresponding to the SARS-CoV-2 viral RNA.

What do results mean for a COVID-19 PCR test?

A positive result happens when the SARS-CoV-2 primers match the DNA in the sample and the sequence is amplified, creating millions of copies. This means the sample is from an infected individual. The primers only amplify genetic material from the virus, so it is unlikely a sample will be positive if viral RNA is not present. If it does, it is called a false positive .

A negative result happens when the SARS-CoV-2 primers do not match the genetic material in the sample and there is no amplification. This means the sample did not contain any virus.

A false negative result happens when a person is infected, but there is not enough viral genetic material in the sample for the PCR test to detect it. This can happen early after a person is exposed. Overall, false negative results are much more likely than false positive results .

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Last updated: January 18, 2022

Polymerase Chain Reaction (PCR)- Principle, Procedure, Types, Applications and Animation

Polymerase Chain Reaction (PCR) is a powerful method for amplifying particular segments of DNA, distinct from cloning and propagation within the host cell. This procedure is carried out entirely biochemically, that is, in vitro. PCR was invented by Kary Mullis in 1983. He shared the Nobel Prize in chemistry with Michael Smith in 1993.

Principle of PCR

PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase adds nucleotides to the 3` end of a custom-designed oligonucleotide when it is annealed to a longer template DNA. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains a region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate its 3` end to generate an extended region of double stranded DNA.

Procedure/Steps of PCR

1. Denaturation

The DNA template is heated to 94° C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA.

2. Annealing

The mixture is cooled to anywhere from 50-70° C. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.

3. Extension

The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.

With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Types of PCR

Real-time PCR
Quantitative real time PCR (Q-RT PCR)
Reverse Transcriptase PCR (RT-PCR)
Multiplex PCR
Long-range PCR
Single-cell PCR
Fast-cycling PCR
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR
In situ PCR
Variable Number of Tandem Repeats (VNTR) PCR
Asymmetric PCR
Repetitive sequence-based PCR
Overlap extension PCR
Assemble PCR
Intersequence-specific PCR(ISSR)
Ligation-mediated PCR
Methylation –specifin PCR
Miniprimer PCR
Solid phase PCR
Touch down PCR, etc

Applications of PCR

PCR is used in analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, anthrax, etc.
PCR can provide information on a patient’s prognosis, and predict response or resistance to therapy. Many cancers are characterized by small mutations in certain genes, and this is what PCR is employed to identify.
PCR is used in the analysis of mutations that occur in many genetic diseases (e.g. cystic fibrosis, sickle cell anaemia, phenylketonuria, muscular dystrophy).
PCR is also used in forensics laboratories and is especially useful because only a tiny amount of original DNA is required, for example, sufficient DNA can be obtained from a droplet of blood or a single hair.
PCR is an essential technique in cloning procedure which allows generation of large amounts of pure DNA from tiny amount of template strand and further study of a particular gene.
The Human Genome Project (HGP) for determining the sequence of the 3 billion base pairs in the human genome, relied heavily on PCR.
PCR has been used to identify and to explore relationships among species in the field of evolutionary biology. In anthropology, it is also used to understand the ancient human migration patterns. In archaeology, it has been used to spot the ancient human race. PCR commonly used by Paleontologists to amplify DNA from extinct species or cryopreserved fossils of millions years and thus can be further studied to elucidate on.

23 thoughts on “Polymerase Chain Reaction (PCR)- Principle, Procedure, Types, Applications and Animation”

What about the tetra ARMS PCR technique?

i just love the way that PCR is explained. its to easy to understand. thanks for this

Best principle of PCR ever explained. Thank you !!!!

I was started working on PCR after finishing my Master, loved the way it produce the accurate and fast result. I was running 3 to 4 test at a time and it gives amazing result. With the help of PCR result it become easy to find how much infection in the blood and based on infection the Specialist doctor can treat the patient and the strength of medicine can be given to cure fastest. Thanks to those people who found this way of doing.

Hi Sir!One important question. How the PCR is working in microbial diagonistics in medical.

specific genetic regions of different microbs are detected and amplified with the help of known primers.

Dear All, I am a medical Microbiologist. I’m interested in Post doctorate fellowship research into genetic studies of Multi-Drug Resistant Tuberculosis. Can somebody link me up. Thanks

how can this polymerase chain reaction be applied in the detection of zika virus in the lab ?

what are u doing explain ur work lil briefly

simply PCR work in 3 steps they are Denaturation where the dubble strand DNA break into single strand. Its temperature is 94 degree Celsius, than annealing temperature where primer bind it temp generally 50-70 degree Celsius and finally extension where the Taq polymerase act on the DNA and primer joining them with hydrogen bonding. simply PCR is the machine which give control temperature, where we prepare our reaction mixture which contain 1.Taq buffer 2.Taq polymerase 3. dNTP which provide nucleotide for the new strands 4.Forward and reverse primer for The reverse primer is designed to attach to the complementary strand to synthesize DNA in the reverse direction — towards the forward primer. The primers are added to PCR experiments to initiate the process of replication by providing the initial nucleotides to the new strand. 5. Template DNA 6. HPLC graded water.

I am working on a bacteria specie with intention of isolating it from different sources and comparing their antibiotic resistance plasmid content and other characteristics.How do I determine the right primers to use for my work?

First i most comment your effort you’re doing great. I am writting to see if anyone can link me to a reputable university where i can run P.hD in molecular microbiology with MRSA as my microorganism of interest. I worked on it in my Masters and i want to continue with it. Am waiting for any assistance one can render.

wat s d role of Thermus aquaticus in PCR and how does it works?

It’s a thermo stable bacterium (Thermus aguatics ) to maintain a temperature duringPCR

An enzyme DNA polymerase is an indispensable tool for DNA replication, we all knows that enzymes are temperature sensitive, Taq DNA is DNA polymerase that’s isolated from bacteria Thermus aquaticus, This enzyme can withstand different temperatures 92,54, 72 degrees.. If during amplification of ur desired DNA (wether Replication or PCR) ur polymerase became inactivated by heat, then the process adding nucleotides and subsequent elongation stops. Therefore Taq DNA is the suitable enzyme to used.

thermus aquaticus is a bacterial specie from which the polymerase enzyme can be obtaines which is use for polymerization in pcr

The taq polymerase used for PCR is from Thermus Aquaticus. And it does not “maintain a tempreature during PCR” but, tolerates and survives in high tempreature, unlike most other bacteria. PCR uses high tempreatures and these polymerases hence work effectively.

Awesome info. Thanks! It would be nice if you share the distinctive futures of the specific types of PCR too. Kind regards

Hi Sir!One important question. How the PCR is working in microbial diagonistics in medical. I need a perfect to present a seminar so plzzz reply me “AS SOON AS POSSIBLE”. “IAM EXPECTING MORE FROM THIS WEBSITE”

1st analyse the microbe DNA and get sequence and then identified the microbe specious then proceed for antimicrobial methods.

dr how can downlaod the books of immunology?

The multiplex endpoint PCR technology offers a number of potential advantages, results are available in a matter of hours rather than days, the extreme sensibility facilitates detection of even minutes the amounts of pathogen DNA in clinical samples. While microbiological culture is likely to remain a gold standard for infection diagnosis, there is growing interest at the potential of PCR technology to provide early, time critical information based on detection and recognition of bacterial or fungal pathogen DNA. By this new modern method, the results are chosen in perquisite for giving adequate antibiotics treatment as early as possible in order to improve the standard of care. Dr. Aurel

How Do Test Characteristics Such as Sensitivity, Specificity, and Percent Agreement Inform SARS-CoV-2 Diagnostic Test Interpretation?

Molecular and antigen tests both have high specificity. However, antigen tests and some molecular tests have lower sensitivity and thus greater potential for false-negative results. 8 , 13 Percent agreement is reported in place of sensitivity or specificity when a nonstandard reference is used to evaluate a new test. 14

EVIDENCE SUMMARY

Diagnostic sensitivity is the ability of a test to identify people who have a disease (i.e., the percentage of those with the disease who test positive). 15 Diagnostic specificity is the ability of a test to identify those without disease (i.e., the percentage of people without the disease who test negative). 15 However, with rapid production of new SARS-CoV-2 tests, analytical test characteristics are often reported initially rather than diagnostic sensitivity. For example, analytical sensitivity corresponds to the smallest amount of SARS-CoV-2 that can be detected, often called the limit of detection. Analytical sensitivity does not necessarily correspond to diagnostic sensitivity. 16 Thus, it is important to evaluate SARS-CoV-2 diagnostic test performance in patients and populations.

A Cochrane review, with limited applicability to clinical settings, included 13 evaluations of four SARS-CoV-2 molecular tests, including ID Now and Xpert Xpress ( Table 2 13 , 17 ) , on 2,255 samples and found an average sensitivity of 95.2% (95% CI, 86.7% to 98.3%) and specificity of 98.9% (95% CI, 97.3% to 99.5%). 13 The range of sensitivity was 68% to 100%. The same Cochrane review included eight evaluations of five antigen tests on 943 samples and found an average sensitivity of 56.2% (95% CI, 29.5% to 79.8%) and specificity of 99.5% (95% CI, 98.1% to 99.9%). The range of sensitivity was 0% to 94%. The antigen test findings have minimal applicability in the United States because the review included no tests with FDA Emergency Use Authorization.

In a university population of 1,098 samples ( Table 2 13 , 17 ) , an evaluation of the Sofia SARS Antigen FIA test, which has FDA Emergency Use Authorization, found a sensitivity of 80.0% (95% CI, 64.4% to 90.9%) and specificity of 98.9% (95% CI, 96.2% to 99.9%) in symptomatic people (n = 227). In asymptomatic people (n = 871), sensitivity was 41.2% (95% CI, 18.4% to 67.1%) and specificity was 98.4% (95% CI, 97.3% to 99.1%). 17

Two large evaluations of the BinaxNOW antigen test, which has FDA Emergency Use Authorization, had different performance results. For symptomatic people older than 10 years (n = 827) at a community testing event in Arizona, the test had a sensitivity of 64.2% (95% CI, 56.7% to 71.3%) and specificity of 100.0% (95% CI, 99.4% to 100.0%). 18 In asymptomatic people older than 10 years (n = 2,592) at the same event, the sensitivity was 35.8% (95% CI, 27.3% to 44.9%) and specificity was 99.8% (95% CI, 99.6% to 100.0%). However, in specimens positive on viral culture, an indicator of infectious virus presence, sensitivity was 92.6% for symptomatic people and 78.6% for asymptomatic people. 18 For people of all ages and symptom status (n = 3,302) at a community testing event in San Francisco, the overall sensitivity was 89% (95% CI, 84.3% to 92.7%), and the specificity was 99.9% (95% CI, 99.7% to 100.0%). 19

The FDA has developed a reference standard for molecular SARS-CoV-2 diagnostic tests and lists analytical sensitivity test comparisons at https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-reference-panel-comparative-data . When a reference standard is not used or is unavailable for molecular and antigen tests with FDA Emergency Use Authorization, positive percent agreement and negative percent agreement are reported instead of sensitivity and specificity. 14 Positive percent agreement is the percentage of total positive tests that are the same when comparing a new test and a nonreference standard. Negative percent agreement is the percentage of total negative tests that are the same when comparing a new test and a nonreference standard. 14 For current antigen tests with FDA Emergency Use Authorization, reported positive percent agreement ranges from 80% to 97.6% and reported negative percent agreement ranges from 96.6% to 100%. 12 , 20

How Does Test Timing in Relation to Symptom Onset Inform SARS-CoV-2 Test Interpretation?

Because viral load decreases after symptom onset, false-negative results are more likely with antigen tests that are performed more than five days after symptom onset. 8 , 12 , 20 – 23

Multiple studies have observed decreasing viral load during the week after onset of COVID-19 symptoms. 21 – 23 Molecular tests are more likely than antigen tests to detect SARS-CoV-2 despite this viral load decrease because molecular tests have higher sensitivity. The instructions of all current antigen tests with FDA Emergency Use Authorization warn of the risk of false-negative results from specimens collected five to 12 days after symptom onset because corresponding antigen levels may fall below the level of detection. 8 , 12 , 17

Validation of molecular and antigen test performance in persons with and without symptoms remains an urgent research need. 13 , 24 , 25 However, increased testing frequency as part of a screening program may compensate for limits in test sensitivity, particularly with antigen tests, and facilitate timely isolation of people who are infectious. 8 , 24 , 26

How Does Pretest Probability of Disease Inform SARS-CoV-2 Test Interpretation?

Pretest probability refers to the estimated likelihood of disease before testing. Pretest probability should be based on a patient's exposure to someone with a confirmed or probable case, signs or symptoms of COVID-19, local or population-specific COVID-19 prevalence, and presence of an alternative diagnosis. 8 , 25 , 27

Exposure to SARS-CoV-2 and COVID-19 Signs and Symptoms . Peak COVID-19 infectiousness occurs at and just before symptom onset. 3 Known or suspected exposure to a person with a confirmed or probable case of COVID-19 increases pretest probability of disease. Signs and symptoms of COVID-19 increase the pretest probability by supporting a clinical diagnosis. The timing of testing after exposure also matters. The incubation period, or time from exposure to symptoms, for COVID-19 ranges from two to 14 days, with a median of 5.1 days (97.5% of patients with the disease become symptomatic by 11.5 days). 28 Based on postquarantine transmission risk modeling, Centers for Disease Control and Prevention recommendations include an option to shorten the standard 14-day quarantine to seven days for patients with a negative SARS-CoV-2 diagnostic test result from a sample collected between five and seven days post-exposure. 29 However, not all jurisdictions have adopted this option.

COVID-19 Prevalence . Disease prevalence affects the predictive value, or the likelihood a person truly does or does not have a disease based on a test result. 8 , 13 Higher disease prevalence increases the predictive value of a positive test result but decreases the predictive value of a negative test result ( Table 2 13 , 17 ) . For example, a negative test result from a resident of a skilled nursing facility where a known outbreak is occurring has a lower negative predictive value because of the high disease prevalence. Likewise, when the pretest probability is low, such as in an asymptomatic individual in a low-prevalence setting, positive predictive value is lower and false-positive results are more common. Clinicians should therefore be familiar with COVID-19 prevalence within populations undergoing testing, as well as seven- to 10-day averages of community disease prevalence as reported by health departments. 8

Alternative Diagnosis . An alternative diagnosis, such as influenza, decreases pretest probability, whereas absence of an alternative diagnosis increases it. 27

How Can Posttest Probability of Disease Be Determined?

A leaf plot provides a visual representation of pre- and posttest probability based on test sensitivity and specificity .

After estimating pretest probability, clinicians must determine the probability of disease based on the test result (posttest probability). Although converting pretest to posttest odds and using likelihood ratios can assist in determining how much to adjust pretest probability given a test result, this approach is cumbersome in practice. Likewise, interpreting a negative result in the context of high pretest probability, or a positive result when pretest probability is low, can be challenging.

A leaf plot offers an alternative through visual representation of pre- and posttest probability based on designated test sensitivity and specificity. 30 Figure 1 shows three leaf plots with the same specificity (98%) but different sensitivities: 70%, 90%, and 99%. To read a leaf plot, the pretest probability is selected on the positive sloped central line (leaf's vein). The conversion to posttest probability with a positive result is the increase in height to the red line. To determine the posttest probability with a negative result, draw a vertical line down to the blue line, and see where it intersects the y-axis. For example, on the leaf plot in Figure 1 with a 90% sensitivity, a 50% pretest probability along the dotted line corresponds to a 10% posttest probability on the blue line in a patient with a negative result. To determine the posttest probability for a positive result, draw a vertical line up from the diagonal to the red line, and see where it intersects the y-axis (in this case, it is approximately 98%). The clinician must judge what threshold of posttest probability determines infection status. 25

What Should Be Considered with an Unexpected Negative Test Result?

A negative molecular or antigen test result might not rule out SARS-CoV-2 infection when pretest probability is high. 13 , 25 , 27 Because false-negative results have implications for disease spread, clinicians should recommend isolation precautions despite a negative test result when pretest probability is high .

Figure 1 shows how the blue curve representing posttest probability with a negative test result progressively lowers with increasing test sensitivity. However, with a high pretest probability of disease, such as 80%, the posttest probability with a negative test result remains approximately 56%, 29%, and 4% with test sensitivities of 70%, 90%, and 99%, respectively. These observations show the need for highly sensitive SARS-CoV-2 diagnostic tests.

Although validation is needed, the Infectious Diseases Society of America suggests that clinicians repeat a SARS-CoV-2 diagnostic test 24 to 48 hours after a single negative molecular test result when pretest probability is high (e.g., symptomatic patient in a hospital setting). 31 Instructions for antigen tests with FDA Emergency Use Authorization advise retesting with a molecular test after an initial negative antigen result when pretest probability is high. 12 The Centers for Disease Control and Prevention recommends that this confirmatory molecular testing occur within 48 hours of the antigen test date. 8

Is a Symptom- or Test-Based Approach Preferred for Discontinuing Isolation Precautions for Most Patients with COVID-19?

A symptom-based approach is preferred in most cases. 32 RT-PCR detects viral RNA, whereas viral culture indicates presence of virus with replication ability and thus potential infectivity. 21 RT-PCR detection of viral RNA does not necessarily correlate with infectivity. Although “prolonged positives” have been detected by RT-PCR for up to 12 weeks, SARS-CoV-2 has not been cultured more than 10 days after symptom onset in patients with mild to moderate COVID-19. 1 , 21 – 23 , 33 , 34

A study of 193 symptomatic and 110 asymptomatic patients with SARS-CoV-2 infection found that viral RNA detection lasted a median of 17 to 19 days. 35 Although viral load peaks near symptom onset and is similar between asymptomatic and symptomatic individuals, the probability of culturing SARS-CoV-2 from the upper respiratory tract decreases as time from symptom onset increases, falling to zero more than 10 days after symptom onset in patients with mild to moderate COVID-19. 3 , 21 , 35 In addition to time after symptom onset, patients should have symptom improvement and no fever for 24 hours without antipyretics before discontinuing isolation. 32

How Should Clinicians Counsel Patients about Serologic SARS-CoV-2 Test Results?

Current SARS-CoV-2 antibody tests detect IgM or IgG to viral spike or nucleocapsid proteins. 11 Antibody tests may help identify past SARS-CoV-2 infection if performed two to four weeks after symptom onset. 36 , 37 Antibody test results should not yet be used to infer immunity to SARS-CoV-2 infection or inform decisions to discontinue social distancing or use of face masks or personal protective equipment. 37

A Cochrane review of 54 studies with 15,976 total samples (8,526 with known SARS-CoV-2 infection) from mostly hospitalized patients found that antibody tests may help confirm past SARS-CoV-2 infection in people who had symptoms more than two weeks before testing. 36 However, the review found few data on the presence of antibodies beyond 35 days after symptom onset. If antibody testing is used, the Infectious Diseases Society of America suggests testing for SARS-CoV-2 IgG or total antibody levels three to four weeks after symptom onset. 37 To assess prior infection in people vaccinated with the Pfizer-BioNTech, Moderna, or Janssen vaccine, an IgM or IgG test to the nucleocapsid protein should be selected because the vaccines encode for the spike protein. 11 Because of current uncertainty about the extent and durability of natural and vaccine-induced immunity, antibody tests are not recommended to determine immune status at this time. 9 , 11 , 37 , 38

Data Sources: A PubMed literature search was completed using the key words SARS-CoV-2 or COVID-19 or leaf plot with test, Cochrane, molecular, PCR, antigen, pretest probability, false negative, sensitivity, viral load, or viral culture. The U.S. Food and Drug Administration, Infectious Diseases Society of America, and Centers for Disease Control and Prevention websites were reviewed. Centers for Disease Control and Prevention sources were cross-referenced in PubMed. An Essential Evidence Plus summary on COVID-19 was reviewed. Search dates: September 17 to October 6, 2020; December 8 to 12, 2020; January 12, 2021; and February 14, 2021.

Arons MM, Hatfield KM, Reddy SC, et al.; Public Health–Seattle and King County and CDC COVID-19 Investigation Team. Presymptomatic SARS-CoV-2 infections and transmission in a skilled nursing facility. N Engl J Med. 2020;382(22):2081-2090.

Cheng HY, Jian SW, Liu DP, et al.; Taiwan COVID-19 Outbreak Investigation Team. Contact tracing assessment of COVID-19 transmission dynamics in Taiwan and risk at different exposure periods before and after symptom onset. JAMA Intern Med. 2020;180(9):1156-1163.

He X, Lau EHY, Wu P, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19 [published correction appears in Nat Med . 2020;26(9):1491–1493]. Nat Med. 2020;26(5):672-675.

Buitrago-Garcia D, Egli-Gany D, Counotte MJ, et al. Occurrence and transmission potential of asymptomatic and presymptomatic SARS-CoV-2 infections: a living systematic review and meta-analysis. PLoS Med. 2020;17(9):e1003346.

Ra SH, Lim JS, Kim GU, et al. Upper respiratory viral load in asymptomatic individuals and mildly symptomatic patients with SARS-CoV-2 infection. Thorax. 2021;76(1):61-63.

Oran DP, Topol EJ. Prevalence of asymptomatic SARS-CoV-2 infection: a narrative review. Ann Intern Med. 2020;173(5):362-367.

Ebell MH, Chupp C, Bentivegna M. A high proportion of SARS-CoV-2-infected university students are asymptomatic. J Fam Pract. 2020;69(9):428-429.

Centers for Disease Control and Prevention. Interim guidance for antigen testing for SARS-CoV-2. Updated December 16, 2020. Accessed February 14, 2021. https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-guidelines.html

U.S. Food and Drug Administration. Coronavirus disease 2019 testing basics. Updated November 6, 2020. Accessed February 14, 2021. https://www.fda.gov/consumers/consumer-updates/coronavirus-testing-basics

Johns Hopkins Bloomberg School of Public Health. Center for Health Security. Molecular-based tests for COVID-19. Accessed November 28, 2020. https://www.centerforhealthsecurity.org/resources/COVID-19/molecular-based-tests/

Centers for Disease Control and Prevention. Interim clinical considerations for use of mRNA COVID-19 vaccines currently authorized in the United States. Updated March 5, 2021. Accessed March 15, 2021. https://www.cdc.gov/vaccines/covid-19/info-by-product/clinical-considerations.html

U.S. Food and Drug Administration. In vitro diagnostics EUAs. Updated March 12, 2021. Accessed March 15, 2021. https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas#individual-antigen

Dinnes J, Deeks JJ, Adriano A, et al.; Cochrane COVID-19 Diagnostic Test Accuracy Group. Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. Cochrane Database Syst Rev. 2020(8):CD013705.

U.S. Food and Drug Administration. Statistical guidance on reporting results from studies evaluating diagnostic tests – guidance for industry and FDA staff. March 2007. Accessed December 12, 2020. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/statistical-guidance-reporting-results-studies-evaluating-diagnostic-tests-guidance-industry-and-fda

Oleckno WA. Epidemiology: Concepts and Methods . Waveland Press; 2008.

Saah AJ, Hoover DR. “Sensitivity” and “specificity” reconsidered. Ann Intern Med. 1997;126(1):91-94.

Pray IW, Ford L, Cole D, et al. Performance of an antigen-based test for asymptomatic and symptomatic SARS-CoV-2 testing at two university campuses - Wisconsin, September–October 2020. MMWR Morb Mortal Wkly Rep. 2021;69(5152):1642-1647.

Prince-Guerra JL, Almendares O, Nolen LD, et al. Evaluation of Abbott BinaxNOW rapid antigen test for SARS-CoV-2 infection at two community-based testing sites - Pima County, Arizona, November 3–17, 2020. MMWR Morb Mortal Wkly Rep. 2021;70(3):100-105.

Pilarowski G, Marquez C, Rubio L, et al. Field performance and public health response using the BinaxNOW TM Rapid SARS-CoV-2 antigen detection assay during community-based testing [published online December 26, 2020]. Clin Infect Dis . Accessed February 14, 2021. https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1890/6052342

Association of Public Health Laboratories. Considerations for implementation of SARS-CoV-2 rapid antigen testing. Version 4. September 24, 2020. Accessed February 14, 2021. https://www.aphl.org/programs/preparedness/Crisis-Management/Documents/APHL-SARSCov2-Antigen-Testing-Considerations.pdf

COVID-19 Investigation Team. Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the United States. Nat Med. 2020;26(6):861-868.

van Kampen JJA, van de Vijver DAMC, Fraaij PLA, et al. Duration and key determinants of infectious virus shedding in hospitalized patients with coronavirus disease-2019 (COVID-19). Nat Commun. 2021;12(1):267.

Wölfel R, Corman VM, Guggemos W, et al. Virological assessment of hospitalized patients with COVID-2019 [published correction appears in Nature . 2020;588(7839): E35]. Nature. 2020;581(7809):465-469.

U.S. Food and Drug Administration. FAQs on testing for SARS-CoV-2. Updated December 10, 2020. Accessed February 14, 2021. https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/covid-19-test-uses-faqs-testing-sars-cov-2

Woloshin S, Patel N, Kesselheim AS. False negative tests for SARS-CoV-2 infection. N Engl J Med. 2020;383(6):e38.

Mina MJ, Parker R, Larremore DB. Rethinking COVID-19 test sensitivity. N Engl J Med. 2020;383(22):e120.

Watson J, Whiting PF, Brush JE. Interpreting a COVID-19 test result. BMJ. 2020;369:m1808.

Lauer SA, Grantz KH, Bi Q, et al. The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application. Ann Intern Med. 2020;172(9):577-582.

Centers for Disease Control and Prevention. Options to reduce quarantine for contacts of persons with SARS-CoV-2 infection using symptom monitoring and diagnostic testing. Updated December 2, 2020. Accessed December 6, 2020. https://www.cdc.gov/coronavirus/2019-ncov/more/scientific-brief-options-to-reduce-quarantine.html

Coulthard MG, Coulthard T. The leaf plot. Br J Gen Pract. 2019;69(681):205-206.

Hanson KE, Caliendo AM, Arias CA, et al. The Infectious Diseases Society of America guidelines on the diagnosis of COVID-19: molecular diagnostic testing [published online January 22, 2021]. Clin Infect Dis . Accessed January 25, 2021. https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciab048/6106562

Centers for Disease Control and Prevention. Duration of isolation and precautions for adults with COVID-19. Updated February 13, 2021. Accessed March 15, 2021. https://www.cdc.gov/coronavirus/2019-ncov/hcp/duration-isolation.html

Li N, Wang X, Lv T. Prolonged SARS-CoV-2 RNA shedding. J Med Virol. 2020;92(11):2286-2287.

Bullard J, Dust K, Funk D, et al. Predicting infectious SARS-CoV-2 from diagnostic samples. Clin Infect Dis. 2020;71(10):2663-2666.

Lee S, Kim T, Lee E, et al. Clinical course and molecular viral shedding among asymptomatic and symptomatic patients with SARS-CoV-2 infection in a community treatment center in the Republic of Korea. JAMA Intern Med. 2020;180(11):1-6.

Deeks JJ, Dinnes J, Takwoingi Y, et al. Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database Syst Rev. 2020(6):CD013652.

Hanson KE, Caliendo AM, Arias CA, et al. Infectious Diseases Society of America guidelines on the diagnosis of COVID-19: serologic testing. August 18, 2020. Accessed February 14, 2021. https://www.idsociety.org/practice-guideline/covid-19-guideline-serology

Infectious Diseases Society of America. Vaccines FAQ. Updated March 5, 2021. Accessed March 15, 2021. https://www.idsociety.org/covid-19-real-time-learning-network/vaccines/vaccines-information--faq/

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COVID-19 diagnostic testing

COVID-19 diagnostic testing shows current infection with the virus that causes coronavirus disease 2019 (COVID-19). The U.S. Food and Drug Administration (FDA) approved the following types of tests for diagnosing COVID-19.

Molecular tests

These tests look for genetic material from the COVID-19 virus.

Polymerase chain reaction tests, shortened to PCR tests, are molecular tests. The lab technique used is called reverse transcription polymerase chain reaction, shortened to RT-PCR. You also may see this type of test called an NAAT test, short for nucleic acid amplification test.

PCR tests are more accurate than the other type of COVID-19 test, called an antigen test. PCR tests may be done at home. But they are more likely to be done by a healthcare professional and processed in a lab.

Antigen tests

These tests look for viral proteins called antigens.

Antigen tests also may be called rapid COVID-19 tests or at-home COVID-19 tests. These tests give a quick result.

Antigen tests are accurate, but they are less accurate than PCR tests. This is especially true if you don't have symptoms. If you take an antigen test and are negative for COVID-19, take another antigen test after 48 hours to check the results.

Tests for more than one virus

An RT-PCR test called the Flu SC2 Multiplex Assay can show any of three viruses at the same time: the COVID-19 virus, influenza A and influenza B. Only a single sample is needed to check for all three viruses.

This test has the same risks of false results as do other COVID-19 tests.

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Why it's done

In the U.S., you might need a COVID-19 diagnostic test if:

You have COVID-19 symptoms, such as fever, cough, tiredness or shortness of breath. If you have symptoms, test right away.
You were exposed to the COVID-19 virus. Wait at least five days after you've been exposed to take a test.
A healthcare professional or your public health department suggests a test.

Testing before you plan to be with someone who has a high risk of serious illness from COVID-19 helps prevent the spread of the COVID-19 virus.

If you've had COVID-19 in the last 30 days and don't have symptoms, you might not need to test even if tests are suggested.

Some people who have COVID-19 don't have symptoms, called asymptomatic. But they can still spread the virus to others. People with no symptoms can be tested.

If people with no symptoms have a positive test result, they should follow guidelines for staying away from others. This helps keep the virus from spreading.

More Information

COVID-19 variant
Coronavirus disease 2019 (COVID-19)

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A COVID-19 diagnostic test can have a false-negative result. This means that the test didn't show you have the virus even though you do. You risk spreading the virus to others if you don't take care, such as wearing a face mask, when you're near other people.

The FDA requires that rapid antigen diagnostic tests catch at least 80% of infections. To lower the risk of false-negative results, the FDA recommends testing more than once.

Rarely, COVID-19 rapid antigen tests can give false-positive results.

False-positive results mean the test results show that you have the virus when you don't. A false-positive is rare and thought to happen in less than 1% of results.

In some cases, a false-positive result could be caused by a manufacturing error. Early evidence suggests that this result may be more common in people with a history of autoimmune disease.

Don't use at-home COVID-19 tests that the FDA has not cleared for use. They may give the wrong results.

How you prepare

In the United States, at-home COVID-19 tests are available. You can buy tests in stores, pharmacies or online. It's useful to have tests before you need them. And it's good to have more than one test in case you need to repeat the test.

The FDA approves COVID-19 tests. On the FDA website, you can find a list of approved tests and when they expire. COVID-19 tests also are available from healthcare professionals, some pharmacies and clinics, or at community testing sites.

When taking a test at home, read the directions that come with the test carefully. Follow the instructions exactly to get as accurate a result as possible.

If you go somewhere to get tested, wear a face mask. Wear a mask even if you don't have symptoms.

What you can expect

COVID-19 tests use a sample of mucus from your nose or throat or a sample of saliva. You might collect the sample yourself for an at-home test. Or a healthcare professional might collect the sample.

Nose or throat swab. Most often, tests use a long nasal swab to get the sample. You or a healthcare professional puts the swab into one nostril at a time and turns it. A healthcare professional might swab the back of your throat to collect the sample.
Saliva sample. Some locations test using spit, called saliva. A saliva sample may be a bit less sensitive than a mucus sample that's taken using a long nasal swab. But a saliva test is easier to do. You spit into a tube several times to provide a sample of your saliva to test. The tube then goes to a lab.

A healthcare provider also may test for other illnesses, such as the flu, that have similar symptoms and that you may have.

Supporting Your Child During COVID-19 Nasal Swab Testing

The purpose of this video is to help children get ready for a COVID-19 nasal swab test. Knowing what to expect may help ease any fears they may have. When children know about the medical test they're about to take, the test is easier for them to take. Children as young as 4 years old can watch this video.

Disclaimer: This material is for your education and information only. This content does not replace medical advice, diagnosis or treatment. New medical research may chance this information. If you have questions about a medical condition, always talk with your health care provider.

Jennifer Rodemeyer, Child Life Program Manager, Mayo Clinic: Hi, I'm Jennifer and I am a child life specialist at Mayo Clinic. My job is to help kids like you prepare for medical tests.

You may have heard there is a virus going around that can make people feel sick. A virus is a germ and it is so tiny you can't even see it.

Some people who get this virus can have a fever or a cough and may feel achy and tired, while some people can have this virus and not feel sick at all. People may get this virus from touching things. That's why it's important to wash your hands often with soap and water. The virus also can spread through a cough or a sneeze. So it's important to always cover your cough or sneeze.

Today, even though you may or may not be feeling sick, we will need to give you a test so we know how to best proceed with your medical care. This medical test will tell us if you have the virus.

When you go to take your test, the health care provider will wear special protective clothing. They wear this clothing to keep themselves and you safe from getting germs. They will wear a mask to cover their nose and mouth and a clear plastic shield to protect their eyes.

The most important thing you can do during your test is to sit perfectly still like a statue. To help make sure you don't move, your parent or caregiver will help keep you still and calm during your test. The health care provider needs to touch the inside of the back of your nose with a long, skinny Q-tip. To do this, you need to hold your chin up, then the health care provider will put the Q-tip in your nose for a short time to collect a sample.

While this happens you may feel like you want to push the Q-tip away, but it's really important to stay as still as possible so the health care provider can finish the test. The Q-tip will be in and out of your nose in a few seconds.

Some kids tell me that counting to 3 or taking a deep breath relaxes them before the test happens, and some tell me they like to hold on to their favorite stuffed animal or blanket. Maybe you have your own way to relax.

Remember that during the test, the most important thing to do is to keep your body perfectly still.

You may have many feelings seeing the health care provider wearing different clothing, but know this person is caring and wants to help you.

Thank you for helping us get this test done, so we know how to proceed with your medical care.

Your COVID-19 diagnostic test result could be positive or negative.

Positive result

This means that you have an infection with the virus that causes COVID-19. Positive test results most often are correct, even if you have no symptoms. You can spread the virus to others even if you have no symptoms.

You'll need to stay away from other people, including others in your home who aren't sick. You can go back to your daily activities when your symptoms have been getting better for at least 24 hours and it's been 24 hours since you've had a fever without taking medicines that lower fevers.

You might still be able to spread the virus even though you feel better. To prevent spreading the virus, wear a mask for five more days. Keep your distance from others. Take another home COVID-19 test if you plan to be around others indoors.

If you get a fever or start to feel worse again, start over with staying home and staying away from others. Repeat the cycle of instructions.

If you have severe symptoms of COVID-19 or a health condition that lowers your ability to fight disease, your healthcare professional may tell you to stay away from other people longer than five days.

Negative result

This means that you likely don't have an infection with the COVID-19 virus. But you may have a false-negative test result. If you have symptoms or were around someone who had COVID-19, stay away from others.

Even if you don't have symptoms, test again after 48 hours. If it's still negative, and you think you have COVID, you can test a third time after another 48 hours. Or you can get a molecular test or call your healthcare professional.

COVID-19 testing: What you need to know. Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/testing.html. Accessed March 5, 2024.
CDC's influenza SARS-CoV-2 Multiplex assay. Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/lab/multiplex.html. Accessed March 8, 2024.
At-home COVID-19 antigen tests: Take steps to reduce your risk of false negative results: FDA safety communication. U.S. Food and Drug Administration. https://www.fda.gov/medical-devices/safety-communications/home-covid-19-antigen-tests-take-steps-reduce-your-risk-false-negative-results-fda-safety. Accessed March 29, 2024.
AT-home OTC COVID diagnostic tests. U.S. Food and Drug Administration. https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/home-otc-covid-19-diagnostic-tests. Accessed March 6, 2024.
Zhang Y, et al. Molecular and antigen tests, and sample types for diagnosis of COVID-19: A review. Future Virology. 2022; doi:10.2217/fvl-2021-0256.
AskMayoExpert. COVID-19 testing. Mayo Clinic; 2023.
Caliendo AM, et al. COVID-19 diagnosis. https://www.uptodate.com/contents/search. Accessed March 11, 2024.
Swab the throat as well as the nose: The debate over the best way to test for SARS-CoV-2. Journal of the American Medical Association. 2023; doi:10.1001/jama.2022.23311.
Preventing spread of respiratory viruses when you're sick. Centers for Disease Control and Prevention. https://www.cdc.gov/respiratory-viruses/prevention/precautions-when-sick.html. Accessed March 11, 2024.
Understanding at-home OTC COVID-19 antigen diagnostic test results. U.S. Food and Drug Administration. https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/understanding-home-otc-covid-19-antigen-diagnostic-test-results. Accessed May 29, 2024.
Gans JS, et al. False-positive results in rapid antigen tests for SARS-CoV-2. Journal of the American Medical Association. 2022; doi:10.1001/jama.2021.24355.
Herbert C, et al. Persistent false positive COVID-19 rapid antigen tests. New England Journal of Medicine. 2024; doi:10.1056/NEJMc2313517.

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Introduction, pcr (polymerase chain reaction).

is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies ( amplicons ).

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What are PCR tests?

PCR (polymerase chain reaction) tests are a reliable and accurate way to diagnose certain infectious diseases , some types of cancer , and certain genetic changes. The tests check for small amounts of genetic material of a pathogen (disease-causing organism) or abnormal cells in a sample of your blood, saliva, mucus, or tissue. The genetic material could be:

DNA , which contains the genetic information needed for a person and most other living things to develop and grow. DNA is copied from one generation to the next.
RNA , which contains information copied from DNA. Many types of RNA help your cells make proteins. Some viruses use RNA instead of DNA to carry their genetic information.

Most viruses and other pathogens contain DNA or RNA.

Unlike many other tests, PCR tests can find signs of disease in the earliest stages of infection. They can also find pieces of a virus you had, after you are no longer infected. Other tests may miss early signs of disease because there aren't enough viruses , bacteria , or other pathogens in your sample, or your body hasn't had enough time to develop an antibody response. Antibodies are proteins your immune system makes to attack foreign substances, such as viruses and bacteria. PCR tests can detect disease when there is only a very small amount of pathogens in your body.

As part of a PCR test, a small amount of genetic material in your sample is copied multiple times. The copying process is known as amplification. If your sample contains pathogens, amplification can make them much easier to detect.

Other names: polymerase chain reaction, rtPCR, reverse transcription PCR, qPCR, quantitative PCR, real-time PCR

How are they used?

PCR tests are used to:

Diagnose certain infectious diseases
Identify certain genetic changes that can cause disease
Find small amounts of certain cancer cells that might be missed in other types of tests

How do they work?

To do a PCR test:

A health care professional will take a sample of blood , saliva, mucus, or tissue.
The sample will contain your own DNA and possibly the DNA of a pathogen or cancer cell.
The sample, an enzyme called polymerase, and certain chemicals are put in a thermal cycler. This machine makes copies of a special segment of DNA through heating and cooling cycles.
The copying process is repeated multiple times. After about an hour, billions of copies are made. If a virus or other pathogen is present, it will be shown on the machine.

Certain viruses, including COVID-19 , are made up of RNA rather than DNA. For these viruses, the RNA must be changed into DNA before copying. This process is called reverse transcription PCR (rtPCR).

What happens during a PCR test?

There are different ways to get a sample for a PCR test. Common methods include blood tests and nasal swabs.

During a blood test , a health care professional will take a blood sample from a vein in your arm, using a small needle. After the needle is inserted, a small amount of blood will be collected into a test tube or vial. You may feel a little sting when the needle goes in or out. This usually takes less than five minutes.

A nasal swab may be taken from the front part of your nostrils (anterior nares). It also may be taken from the back of your nostrils, in a procedure known as a nasal mid-turbinate (NMT) swab, or from the nasopharynx, the uppermost part of your nose and throat. In some cases, your provider will ask you to do an anterior nares test or an NMT swab yourself.

During an anterior nares test , you will start by tilting your head back. Then you or your provider will:

Gently insert a swab inside your nostril.
Rotate the swab in four or five sweeping circles for 10 to 15 seconds.
Remove the swab and insert it into your second nostril.
Swab the second nostril using the same technique.
Remove the swab.

If you only swab one nostril, don't rotate the swab in your nostril, or keep the swab in one area, you may not get a good sample.

If you are doing the test yourself, your provider will let you know how to prepare and package your sample. This often involves placing your swab in a sterile tube, closing it, and sealing it in a special bag. You would then give the specimen bag to your provider or follow the instructions on how to return your specimen for testing.

During an NMT swab , you will start by tilting your head back. Then you or your provider will:

Gently insert a swab onto the bottom of your nostril, pushing it until you feel it stopping.
Rotate the swab for 10 to 15 seconds.

If you only swab one nostril, don't rotate the swab in your nostril, or only keep the swab in one area, you may not get a good sample.

During a nasopharyngeal (NP) swab:

You will tilt your head back.
Your provider will insert a long swab into your nostril until it reaches your nasopharynx (the upper part of your throat).
Your provider will rotate the swab for 10 to 15 seconds and remove it.

If your provider gets enough of a sample from one of your nostrils, they may only need to do one. But they may need to get a sample from your other nostril as well if they had trouble getting the sample from the first side.

Do I need to do anything to prepare for this test?

You don't need any special preparations for a PCR test.

Are there are any risks to a PCR test?

There is very little risk to having a blood test. You may have slight pain or bruising at the spot where the needle was put in, but most symptoms go away quickly.

A nasal swab may tickle your throat or cause you to cough. A nasopharyngeal swab may be uncomfortable and cause coughing or gagging. All these effects are temporary.

Is there anything else I need to know about PCR tests?

PCR tests are considered the best and most effective method for identifying many infectious diseases, including COVID-19 and HPV . Because they often diagnose infections before symptoms start, PCR tests play a very important role in preventing the spread of diseases.

Learn more about laboratory tests, reference ranges, and understanding results .

Allina Health [Internet]. Minneapolis: Allina Health; Nasopharyngeal culture; [cited 2024 May 22]; [about 2 screens]. Available from: https://account.allinahealth.org/library/content/49/150402
American Cancer Society [Internet]. Atlanta: American Cancer Society Inc.; c2024. Tests used on biopsy and cytology specimens to diagnose and classify cancer; [updated 2023 Aug 1; cited 2024 May 22]; [about 2 screens]. Available from: https://www.cancer.org/treatment/understanding-your-diagnosis/tests/testing-biopsy-and-cytology-specimens-for-cancer/special-tests.html
Bettini A, Lapa D, Garbuglia AR. Diagnostics of Ebola virus. Front Public Health. 2023 Feb 23; [cited 2024 May 22]; [about 18 screens].11:1123024. doi: 10.3389/fpubh.2023.1123024. PMID: 36908455; PMCID: PMC9995846.
Britannica [Internet]. Chicago: Encyclopedia Britannic, Inc.; c2024. Reverse transcriptase; [cited 2024 May 22]; [about 9 screens]. Available from: https://www.britannica.com/science/reverse-transcriptase
Centers for Disease Control and Prevention [Internet]. Atlanta: U.S. Department of Health and Human Services; COVID-19: Testing for COVID-19; [updated 2024 Aug 24; cited 2024 Aug 24]; [about 3 screens]. Available from: https://www.cdc.gov/covid/testing/
Cleveland Clinic: Health Library: Diagnostics & Testing [Internet]. Cleveland (OH): Cleveland Clinic; c2024. Blood Tests; [reviewed 2022 Dec 06; cited 2024 May 22]; [about 16 screens]. Available from: https://my.clevelandclinic.org/health/diagnostics/24508-blood-tests
Cleveland Clinic [Internet]. Cleveland (OH): Cleveland Clinic; c2024. COVID-19 and PCR Testing; [reviewed 2021 Aug 24; cited 2024 May 22]; [about 8 screens]. Available from: https://my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing
Enzo [Internet]. Farmingdale (NY): Enzo Life Sciences, Inc.; c2024. What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR?; [cited 2024 May 22]; [about 5 screens]. Available from: https://www.enzolifesciences.com/science-center/technotes/2017/march/what-are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-qpcr?
FDA: US Food and Drug Administration [Internet]. Silver Spring (MD): U.S. Department of Health and Human Services; COVID-19 Test Basics; [current as of 2023 Sep 7; cited 2024 May 22]; [about 4 screens]. Available from: https://www.fda.gov/consumers/consumer-updates/covid-19-test-basics
Khehra N, Padda IS, Swift CJ. Polymerase Chain Reaction (PCR) [Updated 2023 Mar 6; cited 2024 May 22]; [about 5 screens]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK589663/
Mayo Clinic [Internet]. Mayo Foundation for Medical Education and Research; c1998-2024. COVID-19 diagnostic testing: Overview; [cited 2024 May 22]; [about 8 screens]. Available from: https://www.mayoclinic.org/tests-procedures/covid-19-diagnostic-test/about/pac-20488900
Merck Manual Consumer Version [Internet]. Kenilworth (NJ): Merck & Co. Inc.; c2024. Genetic Diagnostic Technologies; [updated 2023 Jun; cited 2024 May 22]; [about 2 screens]. Available from: https://www.merckmanuals.com/home/fundamentals/genetics/genetic-diagnostic-technologies
National Cancer Institute [Internet]. Bethesda (MD): U.S. Department of Health and Human Services; NCI Dictionary of Cancer Terms: DNA; [cited 2024 May 22]; [about 1 screen]. Available from: https://www.cancer.gov/publications/dictionaries/cancer-terms/def/dna
National Cancer Institute [Internet]. Bethesda (MD): U.S. Department of Health and Human Services; NCI Dictionary of Cancer Terms: molecular testing; [cited 2024 May 22]; [about 1 screen]. Available from: https://www.cancer.gov/publications/dictionaries/cancer-terms/def/molecular-testing
National Cancer Institute [Internet]. Bethesda (MD): U.S. Department of Health and Human Services; NCI Dictionary of Cancer Terms: RNA; [cited 2024 May 22]; [about 1 screen]. Available from: https://www.cancer.gov/publications/dictionaries/cancer-terms/def/rna
Nemours KidsHealth [Internet]. Jacksonville (FL): The Nemours Foundation; c1995-2024. Getting a Blood Test; [reviewed 2021 Sep; cited 2024 May 22]; [about 4 screens]. Available from: https://kidshealth.org/en/parents/blood-tests.html
New England BioLabs [Internet]. Frankfurt (GER): New England BioLabs, c2021. qPCR (Real-Time PCR) and RT-qPCR; [cited 2024 May 22]; [about 3 screens]. Available from: https://www.neb-online.de/en/pcr-and-dna-amplification/qpcr-real-time-pcr-and-rt-qpcr
NIH National Human Genome Research Institute [Internet]. Bethesda (MD): U.S. Department of Health and Human Services; Polymerase Chain Reaction (PCR) Fact Sheet; 2020 Aug 17 [cited 2024 May 22]; [about 2 screens]. Available from: https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet
NIH National Human Genome Research Institute [Internet]. Bethesda (MD): U.S. Department of Health and Human Services; Understanding COVID-19 PCR Testing; 2022 Jan 18 [cited 2024 May 22]; [about 4 screens]. Available from: https://www.genome.gov/about-genomics/fact-sheets/Understanding-COVID-19-PCR-Testing
Tan X, Sun L, Chen J, Chen ZJ. Detection of Microbial Infections Through Innate Immune Sensing of Nucleic Acids. Annu Rev Microbiol [Internet]. 2018 Sep 8 [cited 2024 May 22]; 72:447-478. Available from: https://pubmed.ncbi.nlm.nih.gov/30200854
The Free Dictionary [Internet]. Farlex, Inc. c2003-2024. Medical Dictionary: pathogen; [cited 2024 May 22]; [about 1 screen]. Available from: https://medical-dictionary.thefreedictionary.com/pathogen
The International Atomic Energy Agency. How Do COVID-19 Tests Work: RT-PCR Explained [Internet]. YouTube. 2021 Feb 12 [cited 2024 May 22]; Available from: https://www.youtube.com/watch?v=xiTQ6-MOgdk
Thermo Fisher Scientific [Internet]. Waltham (MA): Thermo Fisher Scientific, Inc.; c2024. The Basics: RT-PCR; [cited 2024 May 22]; [about 4 screens]. Available from: https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rtpcr-analysis/general-articles/rt--pcr-the-basics.html
Thermo Fisher Scientific [Internet]. Waltham (MA): Thermo Fisher Scientific, Inc.; c2024. What is qPCR?; 2020 Feb 7 [cited 2024 May 22]; [about 3 screens]. Available from: https://www.thermofisher.com/blog/ask-a-scientist/what-is-qpcr
Vermont Department of Health [Internet]. Burlington (VT): Procedure for Collecting an Anterior Nares Swab; 2020 Jun 22 [cited 2024 May 22]; [about 5 screens]. Available from: https://www.healthvermont.gov/sites/default/files/DEPRIP.EMSNasalNares%20Procedure%20for%20Anterior%20Nares%20Nasal%20Swab.pdf
Verywell Health [Internet]. New York: About, Inc.; c2024. Polymerase Chain Reaction (PCR) for STI Detection and Testing; 2024 Apr 29 [cited 2024 May 22]; [about 6 screens]. Available from: https://www.verywellhealth.com/polymerase-chain-reaction-pcr-3132814

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Interpreting a covid...

Interpreting a covid-19 test result

Read our latest coverage of the coronavirus pandemic.

Related content
Peer review
Jessica Watson , GP and National Institute for Health Research doctoral research fellow 1 ,
Penny F Whiting , associate professor in clinical epidemiology 1 ,
John E Brush , professor of internal medicine 2
1 Centre for Academic Primary Care, Bristol Medical School, University of Bristol, Bristol, UK
2 Sentara Healthcare and Eastern Virginia Medical School, Norfolk, VA, USA
Correspondence to J Watson Jessica.Watson{at}bristol.ac.uk

What you need to know

Interpreting the result of a test for covid-19 depends on two things: the accuracy of the test, and the pre-test probability or estimated risk of disease before testing

A positive RT-PCR test for covid-19 test has more weight than a negative test because of the test’s high specificity but moderate sensitivity

A single negative covid-19 test should not be used as a rule-out in patients with strongly suggestive symptoms

Clinicians should share information with patients about the accuracy of covid-19 tests

Across the world there is a clamour for covid-19 testing, with Tedros Adhanom Ghebreyesus, director general of the World Health Organization, encouraging countries to “test, test, test.” 1 The availability of the complete genome of covid-19 early in the epidemic facilitated development of tests to detect viral RNA. 2 Multiple assays with different gene targets have been developed using reverse transcriptase polymerase chain reaction (RT-PCR). 3 These viral RNA tests use samples usually obtained from the respiratory tract by nasopharyngeal swab, to detect current infections. Serology blood tests to detect antibodies indicating past infection are being developed; these will not be considered in depth in this article.

Testing for covid-19 enables infected individuals to be identified and isolated to reduce spread, 4 allows contact tracing for exposed individuals, 5 and provides knowledge of regional and national rates of infection to inform public health interventions. However, questions remain on how to apply test results to make optimal decisions about individual patients.

Search strategy

This article was produced at speed to address an urgent need to address uncertainties in testing for covid-19. We searched Pubmed using the terms “covid”, “SARS-CoV-2”, “sensitivity”, “specificity”, “diagnosis”, “test”, and “PCR”, and KSR evidence using terms for covid and test. This was supplemented by discussion with colleagues undertaking formal systematic reviews into covid-19 diagnosis.

How accurate are test results?

No test gives a 100% accurate result; tests need to be evaluated to determine their sensitivity and specificity, ideally by comparison with a “gold standard.” The lack of such a clear-cut “gold-standard” for covid-19 testing makes evaluation of test accuracy challenging.

A systematic review of the accuracy of covid-19 tests reported false negative rates of between 2% and 29% (equating to sensitivity of 71-98%), based on negative RT-PCR tests which were positive on repeat testing. 6 The use of repeat RT-PCR testing as gold standard is likely to underestimate the true rate of false negatives, as not all patients in the included studies received repeat testing and those with clinically diagnosed covid-19 were not considered as actually having covid-19. 6

Accuracy of viral RNA swabs in clinical practice varies depending on the site and quality of sampling. In one study, sensitivity of RT-PCR in 205 patients varied, at 93% for broncho-alveolar lavage, 72% for sputum, 63% for nasal swabs, and only 32% for throat swabs. 7 Accuracy is also likely to vary depending on stage of disease 8 and degree of viral multiplication or clearance. 9 Higher sensitivities are reported depending on which gene targets are used, and whether multiple gene tests are used in combination. 3 10 Reported accuracies are much higher for in vitro studies, which measure performance of primers using coronavirus cell culture in carefully controlled conditions. 2

The lack of a clear-cut “gold-standard” is a challenge for evaluating covid-19 tests; pragmatically, clinical adjudication may be the best available “gold standard,” based on repeat swabs, history, and contact with patients known to have covid-19, chest radiographs, and computed tomography scans. Inevitably this introduces some incorporation bias, where the test being evaluated forms part of the reference standard, and this would tend to inflate the measured sensitivity of these tests. 11 Disease prevalence can also affect estimates of accuracy: tests developed and evaluated in populations with high prevalence (eg, secondary care) may have lower sensitivity when applied in a lower prevalence setting (eg, primary care). 11

One community based study of 4653 close contacts of patients with covid-19 tested RT-PCR throat swabs every 48 hours during a 14 day quarantine period. Of 129 eventually diagnosed with covid-19 by RT-PCR, 92 (71.3%) had a positive test on the first throat swab, equating to a sensitivity of 71% in this lower prevalence, community setting. 12

Further evidence and independent validation of covid-19 tests are needed. 13 As current studies show marked variation and are likely to overestimate sensitivity, we will use the lower end of current estimates from systematic reviews, 6 with the approximate numbers of 70% for sensitivity and 95% for specificity for illustrative purposes.

What do clinicians need to know to understand a test result?

Sensitivity and specificity can be confusing terms that may be misunderstood 14 (see supplementary file ‘Definitions and formulae for calculating measures of test accuracy’). Sensitivity is the proportion of patients with disease who have a positive test, or the true positive rate. Specificity is the proportion of patients without disease who have a negative test, or true negative rate. These terms describe the operating characteristics of a test and can be used to gauge the credibility of a test result. They can be combined to calculate likelihood ratios, which are dimensionless numbers that indicate the strength of a positive or negative test result. 15 For calculating probabilities, a likelihood ratio can be used as a multiplier to convert pre-test odds to post-test odds. Positive likelihood ratios greater than 1 are progressively stronger, with 10 representing a very strong positive test result. Negative likelihood ratios less than 1 are also progressively stronger, with 0.1 representing a very strong negative test result. In the case of the nasopharyngeal swab RNA test for covid-19, the positive likelihood ratio is about 14, which is excellent. 6 A positive covid-19 test result should be very compelling. The negative likelihood ratio is 0.3, which is a moderate result, but not nearly as compelling as a positive result because of the moderate sensitivity (about 70%) of the covid-19 test.

Interpretation of a test result depends not only on the characteristics of the test itself but also on the pre-test probability of disease. Clinicians use a heuristic (a learned mental short cut) called anchoring and adjusting to settle on a pre-test probability (called the anchor). They then adjust this probability based on additional information. This heuristic is a useful short cut but comes with the potential for bias. When people fail to estimate the pre-test probability and only respond to a piece of new information, they commit a fallacy called base-rate neglect. Another fallacy called anchoring is failing adequately to adjust one’s probability estimate, given the strength of new information. Likelihood ratios can give a clinician an idea of how much to adjust their probability estimates. Clinicians intuitively use anchoring and adjusting thoughtfully to estimate pre- and post-test probabilities unconsciously in everyday clinical practice. However, faced with a new and unfamiliar disease such as covid-19, mental short cuts can be uncertain and unreliable and public narrative about the definitive nature of testing can skew perceptions.

Figure 1 shows how a clinician’s thinking about a patient’s probability should shift, based on either a positive or negative test result for covid-19. First, the clinician should estimate a pre-test probability, using knowledge of local rates of covid-19 infection from national 16 and regional 17 data and patients’ symptoms and signs, 18 likelihood of alternative diagnoses, and history of exposure to covid-19. After choosing a pre-test probability on the x axis, one should then trace up to either the upper curve for a positive test result or the lower curve for a negative test result, then trace over to the y axis to read the estimate for post-test probability. The figure shows that the shift in the probability is asymmetric, with a positive test result having a greater impact than a negative test result, owing to the modest sensitivity and negative likelihood ratio of the RNA test.

Leaf plot for covid-19 RT-PCR tests based on a sensitivity of 70% and specificity of 95%. The x axis gives the estimated pre-test probability of covid-19 based on the clinical details. The post-test probability is obtained by tracing up and across to the y axis from the lower curve for a negative test, or to the upper curve for a positive test result. The dashed lines illustrate pre-test probability of 90% (clinical case 1) and 50% (clinical case 2)

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The infographic ( fig 2 ) shows the outcomes when 100 people with a pre-test probability of 80% are tested for covid-19 using natural frequencies, which are generally easier to understand. Online calculators are available which allow clinicians to adjust pre-test probability, sensitivity, and specificity to estimate post-test probability 19

Infographic showing outcomes of 100 people who are tested for covid-19

What else should clinicians consider when interpreting test results?

A single negative test result may not be informative if the pre-test probability is high.

A 52 year old general practitioner in London develops a cough, intermittent fever, and malaise. On day 2 of his illness he receives a nasopharyngeal swab test for covid-19, which is reported as negative. His cough and low-grade fever persist but he feels systemically well enough to return to work. What should he do?

Pre-test probability is high in someone with typical symptoms of covid-19, an occupational risk of exposure, and working in a high prevalence region, and negative test results can therefore be misleading. Table 1 shows that for a pre-test probability of 90%, someone with a negative test has a 74% chance of having covid-19; with two negative tests this risk is still around 47%. If this doctor were to return to work and subsequently the test was confirmed as a false negative, then the decision to work would potentially have significant consequences for his patients, colleagues, and everyone with whom he came into contact. It is therefore safest for this GP with strongly suggestive symptoms to self-isolate in line with guidelines for covid-19, even though his test results are negative. This case illustrates the fallacy of base-rate neglect; it can be tempting to trust the results of an “objective” test more than one’s own “subjective” clinical judgement. In general, during this pandemic, pre-test probabilities of covid-19 will be high, particularly in high prevalence secondary care settings.

Pre- and post- test probabilities for covid-19 RT-PCR tests, calculations based on a sensitivity of 70% and specificity of 95%

View inline

A possible alternative diagnosis will reduce the pre-test probability

A 73 year old woman with severe chronic obstructive pulmonary disease (COPD) and a chronic cough develops acute shortness of breath and slight worsening of her non-productive cough. She reports no fever, has no known exposure to covid-19, and no recent travel. She presents to an emergency department where she is acutely short of breath. A chest radiograph shows possible infiltrates in the right upper and middle lung fields. She is admitted and placed in isolation on droplet precautions. She requires intubation for worsening respiratory distress. Initial nasopharyngeal covid-19 testing is negative. Should she remain in isolation on droplet precautions?

This patient has an alternative possible diagnosis: community-acquired pneumonia. Given her lack of other risk factors or clinical symptoms, and chest radiography findings we therefore estimate her pre-test probability at about 50%. One negative test reduces this risk to 24%, the patient therefore has an additional independently sampled nasopharyngeal swab RNA test which was negative, giving a post-test probability after two negative tests of less than 10%. She is treated with antibiotics and continues to recover.

What are the implications for practice and policy?

While positive tests for covid-19 are clinically useful, negative tests need to be interpreted with caution, taking into account the pre-test probability of disease. This has important implications for clinicians interpreting tests and policymakers designing diagnostic algorithms for covid-19. The Chinese handbook of covid-19 prevention and treatment states “ if the nucleic acid test is negative at the beginning, samples should continue to be collected and tested on subsequent days. ” 20 False negatives carry substantial risks; patients may be moved into non-covid-19 wards leading to spread of hospital acquired covid-19 infection, 21 carers could spread infection to vulnerable dependents, and healthcare workers risk spreading covid-19 to multiple vulnerable individuals. Clear evidence-based guidelines on repeat testing are needed, to reduce the risk of false negatives.

Clinicians should ensure that patients are counselled about the limitations of tests ( box 1 ). Patients with a single negative test but strongly suggestive symptoms of covid-19 should be advised to self-isolate in keeping with guidelines for suspected covid-19.

Possible phrases for explaining covid-19 testing to patients

No test is 100% accurate

If your swab test comes back positive for covid-19 then we can be very confident that you do have covid-19

However, people with covid-19 can be missed by these swab tests. If you have strong symptoms of covid-19, it is safest to self-isolate, even if the swab test does not show covid-19

What is the role of serology tests?

Serology tests, which detect immunoglobulins including IgG and IgM, are under development, 22 23 24 with the aim of detecting individuals who have had previous infection and therefore theoretically developed immunity. The time course and accuracy of serology tests are still under investigation, but the same principles of incorporating the test result with the clinical impression applies. False positive serology tests could cause false reassurance, behaviour change, and disease spread. If suitable accuracy can be established, the benefits of these antibody tests include establishing when healthcare workers are immune, helping to inform decisions about the lifting of lockdowns, and allowing the population to return to work. 25

The WHO message “test, test, test” 1 is important from a population perspective; low sensitivity can be accounted for when assessing burden of disease. However RT-PCR tests have limitations when used to guide decision making for individual patients. Positive tests can be useful to “rule-in” covid-19, a negative swab test cannot be considered definitive for “ruling out.”

How patients were involved in the creation of this article

Patients with covid-19 or possible covid-19 were not involved in the writing of this paper for practical reasons

Education into practice

What is the protocol for covid-19 testing in your organisation?

How do you explain covid-19 test results to patients?

Reflect on a recent clinical case of suspected covid-19—what was your estimated pre-test probability? How did this alter with the results of tests?

Author contributorship: JW JB and PW contributed to the conception of the work, JW ran the searches and wrote the first draft of the paper with assistance from JB. PW developed the tools for fig 2 . JB, JW, and PW all contributed to the revised drafts of the paper and approved the final version for submission.

Acknowledgments: The authors would like to acknowledge Jon Deeks for helpful discussions at an early point in writing this article and Richard Lehman for suggestions and comments on a draft of this article.

Competing interests The BMJ has judged that there are no disqualifying financial ties to commercial companies. The authors declare the following other interests: JB has given Grand Rounds talks on medical reasoning and has published a book The Science of the Art of Medicine: A Guide to Medical Reasoning for which he receives royalties. JW has no competing interests to declare.

Further details of The BMJ policy on financial interests are here: https://www.bmj.com/about-bmj/resources-authors/forms-policies-and-checklists/declaration-competing-interests

Funding: JW is funded by a doctoral research fellowship from the National Institute for Health Research. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research, Health Education England, or the Department of Health.

Patient consent: The cases in this article are fictitious and therefore no consent was needed.

Provenance and peer review: Commissioned, based on an idea from the author; externally peer reviewed.

This article is made freely available for use in accordance with BMJ's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by BMJ. You may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained.

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Petherick A

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Understanding Your PCR Nasal Swab Test Results
A positive PCR test does not yield any information about potential immunity. Please be re-evaluated immediately for worsening symptoms such as shortness of breath or lightheadedness. If you have any questions or need to discuss your results further, please call Aftercare at 844.824.8963 Extension 8120.
Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is a laboratory nucleic acid amplification technique used to denature and renature short segments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences using DNA polymerase I enzyme, an isolate from Thermus aquaticus, known as Taq DNA.[1][2] In 1985, PCR was introduced by Mullis and colleagues for which they received a Nobel prize.[3] It is a ...
Polymerase Chain Reaction (PCR)
What is the end result of PCR? The end product of the polymerase chain reaction is a brand new DNA strand with a double-stranded DNA molecule. Q 7. What happens at 72 degrees in PCR? The 72 degrees' temperature is the optimum for Taq polymerase. Once it reaches this temperature, the extension process begins. Taq polymerase works off the ...
Polymerase Chain Reaction (PCR) Fact Sheet
Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and inexpensive technique used to "amplify" - copy - small segments of DNA. Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification ...
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Nucleic acid amplification tests (NAATs), including PCR tests, are more likely to detect the virus than antigen tests. NAATs tests are the "gold standard" for COVID-19 tests. Your sample will usually be taken by a healthcare provider and transported to a laboratory for testing and may take up to 3 days to receive results.
Understanding COVID-19 PCR Testing
Polymerase chain reaction (PCR) is a laboratory technique that uses selective primers to "copy" specific segments of a DNA sequence. COVID-19 PCR tests use primers that match a segment of the virus's genetic material. This allows many copies of that material to be made, which can be used to detect whether or not the virus is present.
Polymerase chain reaction
A strip of eight PCR tubes, each containing a 100 μL reaction mixture Placing a strip of eight PCR tubes into a thermal cycler. The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study.
Polymerase Chain Reaction (PCR)- Principle, Procedure, Types
Polymerase Chain Reaction (PCR) is a powerful method for amplifying particular segments of DNA, distinct from cloning and propagation within the host cell. This procedure is carried out entirely biochemically, that is, in vitro. PCR was invented by Kary Mullis in 1983. He shared the Nobel Prize in chemistry with Michael Smith in 1993.
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Molecular and antigen tests can detect current SARS-CoV-2 infection and are used to diagnose COVID-19 (). 8, 9 Molecular tests, such as reverse transcriptase polymerase chain reaction (RT-PCR ...
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These tests look for genetic material from the COVID-19 virus. Polymerase chain reaction tests, shortened to PCR tests, are molecular tests. The lab technique used is called reverse transcription polymerase chain reaction, shortened to RT-PCR. You also may see this type of test called an NAAT test, short for nucleic acid amplification test.
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There are currently two primary types of COVID-19 tests being used to test patients for COVID-19: molecular tests (also known as nucleic acid, RNA or PCR tests) and rapid antigen tests. The third ...
Polymerase Chain Reaction (PCR)- Principle, Steps, Applications
PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence. The most widely used target nucleic acid amplification method is the polymerase chain reaction (PCR). This method combines the principles of complementary nucleic acid hybridization with those of nucleic ...
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) Introduction PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the ...
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PCR (polymerase chain reaction) tests are a reliable and accurate way to diagnose certain infectious diseases, some types of cancer, and certain genetic changes. The tests check for small amounts of genetic material of a pathogen (disease-causing organism) or abnormal cells in a sample of your blood, saliva, mucus, or tissue.
Interpreting a covid-19 test result
What you need to know. Interpreting the result of a test for covid-19 depends on two things: the accuracy of the test, and the pre-test probability or estimated risk of disease before testing. A positive RT-PCR test for covid-19 test has more weight than a negative test because of the test's high specificity but moderate sensitivity.
Polymerase chain reaction (PCR)
polymerase chain reaction ( PCR), a technique used to make numerous copies of a specific segment of DNA quickly and accurately. The polymerase chain reaction enables investigators to obtain the large quantities of DNA that are required for various experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and ...
PDF Experiment (5): Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR), a process conceived by Kary Mullis in 1983. (3)It is a laboratory version of DNA replication in cell where particular piece of DNAcan be. amplified in billions of copies in a short time. The PCR amplify a precise fragment of DNA from a complex mixture of starting material termed the template DNAwhich controlled ...

Understanding your PCR nasal swab test results

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Polymerase Chain Reaction (PCR) : Principle, Procedure, Components, Types and Applications

Principle of PCR

Components of PCR

Procedure of PCR

Types of PCR

Applications of PCR

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Q 2. What happens in a polymerase chain reaction?

Q 3. What is needed for PCR?

Q 4. How is the PCR used to diagnose?

Q 5. Is real-time PCR quantitative?

Q 6. What is the end result of PCR?

Q 7. What happens at 72 degrees in PCR?

Q 8. How many types of PCR are there?

Q 9. How accurate is a polymerase chain reaction?

Q 10. What diseases can PCR detect?

Q 11. What do PCR primers do?

Q 12. Why is PCR important?

Q 13. What enzyme is used for PCR?

Q 14. What are the 4 steps of PCR?

Q 15. Who first got the idea of a polymerase chain reaction?

Q 16. Why it is called real-time PCR?

Q 17. Is the RT PCR expensive?

Q 18. What is the difference between real-time PCR and PCR?

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How Do Test Characteristics Such as Sensitivity, Specificity, and Percent Agreement Inform SARS-CoV-2 Diagnostic Test Interpretation?

EVIDENCE SUMMARY

How Does Test Timing in Relation to Symptom Onset Inform SARS-CoV-2 Test Interpretation?

How Does Pretest Probability of Disease Inform SARS-CoV-2 Test Interpretation?

How Can Posttest Probability of Disease Be Determined?

What Should Be Considered with an Unexpected Negative Test Result?

Is a Symptom- or Test-Based Approach Preferred for Discontinuing Isolation Precautions for Most Patients with COVID-19?

How Should Clinicians Counsel Patients about Serologic SARS-CoV-2 Test Results?

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