The Real-Time Reverse Transcriptase-Polymerase Chain Reaction Test (rRT-PCR) is intended for the qualitative detection of nucleic acid from COVID-19, either from upper or lower respiratory tract specimens . The PCR test results then determine if a person has contracted COVID-19 or not.
The PCR test is considered the “gold standard” test for diagnosing COVID-19 because it is considered the most accurate and reliable type of test[1]. In this article, we will delve deeper into RT-PCR testing—the CT value and its meaning in PCR.
Principles of rRT-PCR Testing
The rRT-PCR test supports the detection of specific regions in the coronavirus nucleocapsid gene and human RNase P with the help of primers and probes.
First of all, viral RNA is converted into complementary DNA with the help of the reverse transcriptase enzyme. Then DNA polymerase is utilized to build the 2nd strand of complementary DNA to double-stranded DNA.
Finally, this double-stranded DNA is amplified with the help of a PCR machine. PCR tests are the most sensitive and specific tests to detect coronavirus even when few copies of coronavirus are generated with a PCR machine [2]. This is how PCR test results are obtained from a laboratory.
Specimen for rRT-PCR Test
Listed below are the different specimens recommended for RT-PCR testing:
- Nasopharyngeal or oropharyngeal swabs
- Sputum
- Respiratory tract aspirate
- Bronchoalveolar lavage
- Nasopharyngeal wash
These samples are collected from individuals with suspected COVID-19 infection. Aspirates and lavage are managed by healthcare workers. PCR test results will then determine if the person is COVID-19 positive or negative.
How is the Testing Performed?
A sample is first taken from the upper or lower respiratory tract. Viral RNA is extracted from the sample. Then, reverse transcriptase and DNA polymerase are used sequentially to generate double-stranded DNA from viral DNA.
Subsequently, millions of copies of this DNA are made in the PCR machine, also “called thermal cycler”, with different chemicals, enzymes, and nutrients necessary for amplifying DNA. One such chemical is necessary to produce fluorescent light if coronavirus is present in the sample.
Accuracy and Turnaround Time of RT-PCR Covid Tests
Accuracy may vary based on the test. Generally, the accuracy of the rRT-PCR Covid test is 97.2%. The test is considered a gold standard diagnostic test with a minimum turnaround time of about 24 hours, given that the specimens need to be sent to laboratories.
What Do COVID-19 PCR Test Results Mean?
The COVID-19 PCR test result may be positive, negative, false-positive, or false-negative.
- A positive COVID-19 PCR test result means that, most probably, a person may be suffering from coronavirus. Most of the time, individuals with positive COVID-19 PCR test results only suffer from mild symptoms and recover without any specific medical treatment;
- A negative COVID-19 PCR test result means that a person is not infected with the virus;
- A false-positive PCR test result indicates that an individual does not have an infection. Still, the test was positive due to some laboratory or personal error;
- False-negative PCR test result means that an individual may have symptoms of COVID-19, but the PCR test fails to detect the virus due to laboratory errors.
It is also possible that the PCR test result may come out negative when an individual is actually infected. This scenario happens mainly at the start of the infection.
What Does “CT” Mean?
The “CT value” (Cycle Threshold) refers to the highest cut-off value of amplification cycles to generate minimum copies of DNA during the PCR assay that can be detected fluorescently. Let us explain…
During PCR tests, a small amount of DNA in the sample is copied “x” amount of times. Through this amplification process, sample pathogens are also reproduced. With more genetic material at our disposal, it is easier to detect the virus. In this context, the CT value indicates how many cycles are required before the virus can be detected.
When rRT-PCR is performed, a specific number of amplification cycles are performed to make enough copies of the DNA to be detected easily with the help of a fluorescent nucleic acid probe that is fluorescently labeled DNA oligonucleotides. Then, the PCR test results are interpreted to find out if an individual has got the virus or not.
Moreover, CT values may be used to detect how much coronavirus material is present in a sample that has been tested positive.
CT Value Role in Real-Time PCR Test Interpretation
The CT value is determined by the assay manufacturers or laboratories when they are checking for the validation of laboratory-developed PCR test results.
Conclusion
PCR tests play a crucial role in controlling and preventing the spread of COVID-19. Wondfo Biotech is a leading POCT solutions provider whose contribution to offering reliable COVID-19 testing solutions has been remarkable.
Since the beginning of the pandemic, Wondfo Biotech has provided innovative COVID-19 testing solutions to healthcare centers and communities in 140 countries and regions worldwide. With over three decades of experience in the healthcare industry, Wondfo has been committed to the development of PCR tests, rapid antigen tests, and antibody tests.
Hopefully, this article shed some light on PCR testing and some of its characteristics. To know more about Covid-19 PCR testing, visit Wondfo’s official website.
[1] https://www.yalemedicine.org/news/which-covid-test-is-accurate
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