Let's examine RT-PCR
Between February and April, as active cases kept mounting, the world faced a shortage of COVID-19 RT-PCR testing kit components, right from swabs and buffer to enzymes. In the US, Colorado received 7 per cent of the swabs it had requested from the federal government till April 24. California received 90,000 of the 350,000 swabs it had asked for. To maximise the available swabs, CDC revised its guidelines on March 9 and asked technicians to collect one specimen swab instead of two.
A major problem with RT-PCR tests is that it depends on proprietary ingredients, protected by a registered trade name. These canot be quickly developed by other manufacturers to meet the shortfall. Although other versions of the ingredients might work, it’s not easy to simply switch to a different type as even tiny changes
4 The viral RNA is converted to DNA using an enzyme, reverse transcriptase, which induces the process of reverse transcription.This step is needed as only DNA can be amplified. can make the test fail. False results can be disastrous in this fight against COVID-19. In March-end, the Netherlands faced shortage in reagents. It asked Roche, which supplies to most Dutch labs, to share the recipe for its buffer solution. Under pressure from the European Commission, Roche agreed but shared a generic recipe available in text books. That month US also saw short supply of extraction reagent developed by Dutch company Qiagen.
“We will never have enough testing as reliance is on proprietary reagents. There needs to be more sources of PCR reagent, including from domestic producers,” says Leena Menghaney, lawyer with humanitarian group Médecins Sans Frontières.
To overcome the shortage, researchers have come up with alternatives. One USbased Formlabs is working on 3D printing with pliable resin, which can replace the nylon swabs. Others are working on testing methodologies that simply would not require swabs. To reduce the dependence on buffer solution, dry swabs are a) Denaturing or opening DNA helix to single strands.This is done at 95oC b) Primers pick up the needed nucleotides and initiate the process of replication.This is called annealing and done at 55-65oC c) With the help of enzyme polymerase and nucleotides, copies of the strand are made at 72oC and called extension step. d) The process is repeated around 40 times to get a few billion copies of the viral genetic material. 6 Primers have fluorescent probes or dyes which are released after each cycle of replication
also being developed. Some have found that a saline solution or standard buffer solution work equally well as the specialised ones. But these need to be validated by regulatory agencies before they are put to use. Any glitch can further reduce the reliability of RT-PCR, already fraught with accuracy issues. An analysis of the available RT-PCR kits for COVID-19 shows some have sensitivity of just 90 per cent and specificity of 96 per cent. In real world conditions, this could be just 66 to 80 per cent, which means one in every three would be falsely tested as negative.