Increased tracing, testing and isolation of people with novel coronavirus infection is an effective way to contain the virus spread. Currently, the preferred protocol for testing uses the RT-PCR (Reverse Transcription-Polymerase Chain Reaction) test . This protocol does, however, take time and is expensive.
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With the steeply rising number of infected persons, there is a need for a reliable test that would give results quickly and also be less expensive. Researchers from the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, have studied such a method, also using RT-PCR but with dry swabs, bypassing the RNA isolation stage, which they find consumes less time and is less expensive.
They also suggest a variant method which apparently shows a higher efficiency that the conventional one. The results have been posted in bioRxiv and medRxiv preprint servers. Preprints are yet to be peer-reviewed and published in scientific journals.
Variant method: Steps involved in the testing protocol studied by CCMB.
In the usual method of testing, nasal swabs collected from a person are placed in a viral transport medium (VTM). From this, a part of the liquid is taken, the viral RNA is extracted and RT-PCR test is carried out. The remainder is stored. It is the step of isolating the RNA that takes time and is expensive. So, the authors have proposed an alternative method.
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Dry swabs
Instead of placing the nasal swabs in the VTM, they are put in a Tris-EDTA (TE) buffer solution, protected by ice. “Virus in dry swabs can stay for several days at 4 degree [ice temperature]. For longer storage, it can be kept in minus 80 degree,... it [dry swab] is much more suitable than VTM, and testing can be delayed, if necessary,” says Rakesh Mishra from CCMB, and one of the authors of the preprint. He adds that handling and transporting dry swabs is safer and more convenient.
A small part of the dry swab-TE extract was taken in a new vial and heated to 98 degree C. This destroyed the protective wall of the virus particles, releasing its RNA and this was sent for the RT-PCR test. In all, 40 patients were put through both testing protocols (heated TE without RNA extraction and current standard method). While 22 tested positive and 18 negative in the new method, the standard method yielded 23 positives and 17 negatives. The researchers found that the new protocol of using dry swab-TE extract for RT-PCR was at par with the standard method.
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However, the standard method is known to have a problem of false negatives. To address this, the researchers took the dry swabs-TE extract, extracted RNA from it and studied the samples. In this variant method, they found that a few samples that were consistently negative in both methods now showed a positive result. So out of 40 samples they now had 28 positives and 12 negatives. This result was reproduced on testing multiple times. They surmise that this is because of low viral load, which was picked up in the new variant method.
Handy improvisation
“This is an improvised method, makes the COVID-19 testing rapid and less expensive. Both are welcome considering limited availability of reagents for VTM and RNA isolation,” says L.S. Shashidhara, from Ashoka University, Delhi, who was not involved in the research. “This is a good development as we need to increase the number of tests considerably across India. More and more new labs are being enrolled as testing centres and large numbers of people are being trained in RT-PCR-based testing. Fewer the number of steps, fewer would be the errors,” he adds.
Further study is needed to see if the samples collected and stored in TE can be used for culturing of the virus for any future studies and full sequencing of the viral genomes.
