Understanding the Laboratory Testing for novel Corona Virus Disease (COVID-19)

The purpose of this write up is to clarify certain aspects of laboratory testing for Corona Virus.

There are two basic methodologies applied to test for the novel Corona virus (SARS-CoV-2). The first is by detecting the virus itself in the sample, and second is by detecting antibodies in our blood/serum that are developed in response to the viral infection. 

Testing for Virus (Molecular testing): It looks for its RNA.

The SARS-CoV-2 is an enveloped, single-stranded RNA virus, belonging to the family of Coronaviridae. The length of its RNA  is approximately 30 kilo bp (30,000 base pairs). For testing the virus, we need to pick a region (in its genome/ RNA) that is specific for this particular virus, as there may be similarities with other viruses, especially with its own family. Thus, testing is directed at detecting two genes on the viral RNA strand that makes it specific. 

For a positive test, both genes need to be detected. 
The testing procedure for the virus is not easy, as the RNA that is isolated from the sample is unstable and does not stay for long. Further, for it to be detected, it has to be multiplied many times to bring to such a level (amount) that it can be detected by the methods employed. 

  • RT-PCR:  Hence, for all RNA molecules, the first step in testing is to convert it to DNA, which is done by a process using enzyme ‘reverse transcriptase’. Once transcribed back to DNA, it is then amplified using specific primer (short strands of DNA which will flank the region of interest on both sides) by PCR (polymerase chain reaction). Thus, we arrive at the test terminology – RT-PCR.  This is a simple test, inexpensive and can be carried out by anybody who has sufficient experience in carrying out molecular tests. The test, however, is not very quick, and is labour -intensive. It also requires an additional step, after PCR, for detecting the DNA. The test may be negative if the quantity of RNA (or viral load) is not much.
  • This brings us to rRT-PCR (or RT-RT-PCR, or real-time reverse transcriptase PCR), which is a modification of the RT-PCR to make a quantitative estimate of the RNA load, in real time, as the detection process is automated, quick and very sensitive. The real time PCR has an inbuilt system of amplification and detection of DNA using fluorescent signals. The test runs in a machine that continues to amplify the DNA, and once it reaches a threshold, there is fluorescence detected on the computer monitor. Thus, the earlier the detection (i.e. taking lesser number of PCR cycles) the higher the viral load. This method is quicker and can be employed for mass testing, but requires the equipment which is not available in all the laboratories.
  • A third method of detecting the viral RNA, via DNA is by Next Generation Sequencing, which is nothing but ‘multiplexing’ of all the PCRs (one PCR per fragment of DNA is done), in order to cover vast regions of the viral genome. This is also very sensitive, but requires expensive equipment.

Existing Multiplex PCR kits with respiratory virus panels, such as those manufactured by Biofire or Genmark,  DO NOT detect CoV-2, the virus that causes COVID-19.

  • A Fourth test is called a Nucleic Acid Test (NAT): this  is non-PCR automated assay that utilizes isothermal nucleic acid amplification technology for the qualitative detection of SARS-CoV-2 viral nucleic acids in direct samples, be it nasal or nasopharyngeal or respiratory secretions from a suspect case. The SARS-CoV-2 nucleic acid is generally detectable in respiratory specimens during the acute phase of infection. 

All the molecular methods employed for detection of RNA need to be done in tissue which is most likely to harbour the RNA, and thus not necessarily blood. The test may be falsely negative if only upper airways are tested.

Testing for body’s immune response (Serological tests). This method is more wide spread (in general terms) and thus better understood, by doctors and lay persons alike. The test depends upon the presence of the SARS-CoV-2 specific antibody (monoclonal antibody) in the particular sample, mostly serum or plasma of the individual. Hence, this is not preferred in the early stages of the disease, as the body takes some days to mount its immune response. Testing process is simple, usually automated and quick, and inexpensive. The major advantage of this test is that it can detect antibodies for a long time after the infection is over.

Above matter drafted on request by Dr Sunita Bijarnia Mahay, Senior Consultant Geneticist Sir Ganga Ram Hospital, Delhi

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