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Tweaking RT-PCR kits to detect new strains

Date : 28 Feb, 2021


Business Standard I 28 Feb, 2021: With mutations of the Sars-CoV-2 virus on the rise, the makers of reverse transcription polymerase chain reaction (RT-PCR) test kits are now working on adapting them to be more sensitive and detect the mutant strains from samples. The country’s apex health research body, Indian Council of Medical Research (ICMR), is receiving proposals from kit makers in this regard.


ICMR Deputy Director-General Nivedita Gupta told Business Standard, “RT-PCR manufacturers have started coming with differentiated kits. There is a World Health Organization (WHO) advisory on this saying that there are some kits that miss the S-gene (spike protein), and then we know that we have a mutant strain.”


She explained that in case of the UK variant, the concurrence of missing the S-gene in the laboratory and the sample being a UK-variant of the COVID virus is 92-93 per cent.


Pune-based Mylab, which came out with an indigenous RT-PCR kit in March last year, said that its current assay or test has already covered most dominant strains, “We are working on a discriminative test that will enable us to differentiate one variant from the other,” said a company spokesperson. He added that so far Mylab has not received any note from ICMR to start tweaking its test kit.


An Indian kit maker, while asking not to be named, pointed out that making alterations is not difficult, but it has to make commercial sense. “There is not much demand for differentiated RT-PCR kits yet.”


Meanwhile, Transasia, which is all set to start a kit making plant, is working on improving the sensitivity of its kit from the very beginning.


Suresh Vazirani, CMD, Erba-Transasia Group, said, “Current RT-PCR tests are capable of detecting mutants, but to be able to catch all the mutant strains we need to make the tests more sensitive. We are doing that already and will launch it soon.” “Most kits now need 1,000 copies of the DNA to be able to detect the virus. If we don’t want to miss any mutant, we need to bring this down to 100 copies.”, he added.


ICMR has already said that it has found two new variants: N440K and E484Q. The US Food and Drug Administration has asked test markers recently to check sequence alignment of their primers and probes for RT-PCR kits from time to time with the publicly available genome sequence of the SARS-CoV-2 virus.


Routine monitoring is recommended to evaluate what impacts the mutations will have on the test kits.


Gupta said ICMR has never advocated a single gene-based testing kit. “The idea is to have multiple gene targets in an RT-PCR, which is a safety net to pick up different strains. Can an RT-PCR kit theoretically miss a mutant strain?


The current RT-PCR tests are using probes that are based on the genome sequencing of the original Wuhan strain of the virus, said Tony Jose, co-founder and CEO of Clevergene, a deep tech company offering genomics services for contract research and genetic diagnostics.


“A lot of mutations have happened since. Some mutations may have happened on the RT-PCR probe binding site. We can have more than one probe in a test kit to detect any mutant strain. If the RT-PCR test misses a mutant strain, then a COVID-19 positive person may have a negative test result.” Jose said.


Clevergene has signed a memorandum of understanding with the Council of Scientific and Industrial Research and is working on identifying the mutant strains (through genome sequencing) that may have adverse effects on vaccines or on current RT-PCR tests.


Gupta, too, admitted that the sensitivity of the RT-PCR test- the gold standard for COVID-19 testing is 70%. “So even the best test is 70% sensitive and there is obviously a chance of missing a COVID-19 positive case for various reasons.”


ICMR, however, is confident that the testing situation in India, in the backdrop of the mutating strains, is not worrying at the moment as most laboratories are using two or three gene target tests.