By Dr M S Mustak
Aug 19: The novel coronavirus disease has infiltrated into India; hitherto, over two million cases have been reported from the country. Amid the ongoing COVID-19 pandemic, India has witnessed a massive surge of cases in the past 2 weeks. Apart from the nationwide lockdown, India has increased its testing rate and has markedly strengthened the health care sector to combat COVID-19.
Currently, the people suspected with Corona infection undergo testing and came up with the results in mystifying form, i.e., "Positive" or "Negative". It is required that every person that undergoes testing for corona need to know at what measure he or she stands positive or negative. Keeping in view the ambiguity, let's understand how a novice can comprehend results from real-time reverse transcriptase-polymerase chain reaction (commonly known as rRT-PCR test).
Before proceeding towards the depth of the test, it is pertinent to understand the genomic architecture of COVID. The genome of coronavirus is 30,000 “letters” long; which is tiny, when comparing it with human genome which is made up of 3 billion letters. Scientists have identified genes for as many as 29 proteins, which carry out a range of jobs from making copies of the coronavirus to suppressing the body’s immune responses. The first viral protein created inside the infected cell is ORF1ab, is actually a chain of different proteins joined together. Two of these proteins act like scissors, snipping the links between the different proteins.
Along with ORF1ab, there are structural proteins in the virus: Spike protein (commonly recognized as S protein), encoded by S gene; form prominent spikes on the surface of the virus by arranging themselves in groups of three. Part of the spike can extend and attach to a protein called ACE2 (in humans), which appears on particular cells in the human airway.
Envelop protein (common recognized as E gene) form the oily envelop around of the virus, researchers has found that E protein has a role in switching on and off the human genes, once the virus invades.
Nucleocapsid Protein (commonly recognized as N gene) protects the virus RNA, keeping it stable inside the virus by wrapping and coiling.
RNA-dependent RNA polymerase (RdRP gene), RdRP replicates the virus’s genetic material inside the host cell, afterward used to produce a bunch of proteins. The new genetic material and proteins then coalesce into new viral particles that ooze out from the host cell, ready to infect neighbouring cells.
There are several other genes that are observed in the genome of coronavirus. The genes stated above are mainly targeted in the rRT PCR testing and choice of the gene may differ from one manufactures kit to other manufacturers’ kit.
The rRT-PCR test is a gold standard method to diagnose the presence of SARS-CoV-2 in a sample. If SARS-CoV-2 is present in a sample, it means the person from whom the sample was obtained likely has COVID-19.
For the detection of COVID-19 in an individual, samples are taken from the nasopharyngeal (obtained from a person by a swab of the upper respiratory tract) collected in viral transport medium. RNA is then extracted and afterwards, converted to complementary DNA, or cDNA, using an enzyme called reverse transcriptase.
The diagnostic panel for COVID-19 comprises of four target genes. Three genes are specific to the new coronavirus and one is a human gene, used as an internal control. Two synthesized molecules (comprised of either of the genes stated above) are attached to each cDNA strand, a primer and a probe. The primer is a short stretch of nucleotides that binds itself to a location on the cDNA.
The coronavirus-specific genes include a universal gene that codes for the envelope protein, which is found in all coronaviruses. Two other genes, ORF-1a and RdRp, are to be found only in SARS-CoV-2. So finding only the envelope protein gene would mean the virus is a coronavirus, and finding ORF-1a and RdRp would indicate that the coronavirus is SARS-CoV-2.
If the rRT-PCR test is able to detect the presence of all genes targeted for the detection after thermal cycles in the cycler, irrespective of the human control gene, the patient is presumptively said to have tested positive for the new coronavirus and, if not the individual is said to have tested negative for coronavirus. If only one or two genes are detected but not the third, the test is marked as ‘inconclusive’ and has to be repeated for the given conclusive results. There is no possibility of giving positive for COVID-19 suppose a person is infected with common coronavirus that causes common cold/respiratory illness.
Thus, rRT-PCR analysis considered as the gold standard for COVID-19 testing does not give false positive or false negative results. The only thing is for the analysis to be performed in BSL 3 (Bio-safety level 3) lab by well-trained personnel and training taking all precautionary measures.
Dr M S Mustak works as an associate professor in the department of Applied Zoology, Mangalore University.