The Biology Behind RT-PCR and Rapid Test Kits for Covid-19 Detection

Student Contributor: Students of UG 3^rd year Biochemistry

 Presently, in the battle with Covid-19, we need an enormous number of diagnostic tests to be performed on suspected populations to flatten the epidemic curve. Time demands to perform tests and detect more numbers of infected people so that they can be quarantined and in turn, the infection can be checked. World Health Organisation (WHO) has already recommended two tests for the diagnosis of the Covid-19 disease. Among them, the first one and the most reliable testing method is RT-PCR based detection of viral RNA in the swab samples of suspected persons. There is a limitation of the RT-PCR test as it takes two to three days to get the results. As the virus is spreading at higher rate early detection is now very important to prevent the spreading and this is where the other recommendation from WHO comes handy which is much quicker in producing results than RT-PCR. The rapid test kit based on the detection of antibodies to SARS CoV-2 is the other diagnostic tool that we can use for the detection of Covid-19 infected people. The test takes a very short time and we can check the utmost range of individuals with it. Although the rapid test which is nothing but an immunodiagnostic test is not that accurate like the RT-PCR based test it can provide early and quick detection. But one factor must be remembered that the rapid test isn’t the final method to verify the Covid-19 infection. There are question marks regarding the accuracy of rapid tests as IgM and IgG antibodies are generally detectable in the blood after several days (10-14) of Covid-19 infection. Hence,  for confirmation, one needs to perform the RT-PCR test. Here we are going to discuss the biological background associated with both the test the RT-PCR test and the rapid test kits.

Reverse transcriptase is an enzyme that makes RNA dependant DNA copies and polymerase chain reaction or PCR is a thermal technique based on which DNA molecules can be duplicated using sequence-specific primers. RT-PCR based diagnostic kit uses a throat or nasal swab of a suspected person and these samples are first transported to laboratories in ice-cold conditions. Then in the lab, the viral RNA present in the sample is reverse transcribed to DNA using reverse transcriptase enzyme and then the DNA is then amplified using specific sequences of DNA i.e. primers having complementarities to viral sequences. This PCR consists of 30-40 cycles in which exponential amplification of the viral genome can be detected using a chemical signal. The test is highly specific and sensitive as it uses primers designed based on viral genome sequences. The full genome sequence of the SARS-CoV2 virus has been made available on January 10, 2020, in Global Initiative on Sharing All Influenza Data (GISAID) platform and this genome data has helped immensely to design effective primers for RT-PCR test kits. But there are also limitations as RT-PCR is a bit time consuming and requires people with scientific expertise.

The COVID-19 Rapid test is a qualitative lateral flow immunochemical assay for the detection of IgM and IgG antibodies simultaneously for SARS-CoV-2 in blood, body fluid, or plasma specimens. The test cassette contains recombinant SARS-CoV-2 substance conjugated to colored particles. Once a specimen is added to the sample well of the cassette, any IgM and IgG present in the specimen can bind to the antigen conjugate and it forms colored coronavirus antigen-antibody complexes. This mixture moves laterally on the membrane to the test region. In this test region, anti-human IgM and anti-human IgG are immobilized onto the membrane. This captures any IgM and IgG complexes that have formed which causes the appearance of colored lines.

Therefore, if the sample contains SARS-CoV-2 IgM antibodies, a colored line can be seen within the IgM test line region. If the specimen contains SARS-CoV-2 IgG antibodies, a colored line can be observed in the IgG test line region. To serve as an internal control, a colored line will always appear in the control line region, indicating that the proper volume of specimen and buffer has been added, and the proper procedure has been followed. If the test kit is faulty the internal control will not produce any color. So, in that case, whichever line shows the color the test result should not count.

(A) If only the control line appears on the strip it means the sample is negative.

(B) If IgM is detected more than IgG that means the patient could be in the early stage of infection.

(C) If IgG is detected more than IgM that means a patient could also be in a late or recurrent phase of infection.

(D) If each of them is detected in the same quantity that means the patient is within the active phase of infection.