The Indian Council of Medical Research (ICMR) has recommended only a reverse transcription-polymerase chain reaction (RT-qPCR) test for novel coronavirus testing. Researchers at the Centre for Cellular and Molecular Biology (CCMB) have developed a new low-cost and low-tech required test for SARS-CoV-2 testing. This test is known as reverse transcription nested PCR (RT-nPCR) test.
This test does not require real-time quantitative RT-qPCR. The RT-nPCR developed by the CCMB research team has shownÂ comparable performance to the standard RT-qPCR test. The nested PCR (RT-nPCR) approach does not depend on RT-qPCR but uses standard RT-PCR as part of an endpoint assay.
In the course of comparing the results of both tests, researchers found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario, which may be due to low viral representation in many samples. This finding brought home the importance of monitoring detection efficiency directly in test environments.
â€œWe developed and tested an RT-nPCR protocol comprising a multiplex primary RT-PCR for amplification of four SARS-CoV-2 amplicons and a control human amplicon followed by a secondary nested PCR for individual amplicons. We also examined the use of RT-nPCR in pooled testing and in direct amplification without RNA isolation,â€ said Dr Rakesh Mishra, Director, CCMB, while speaking withÂ India Science Wire.
The RNA isolated from nasopharyngealÂ swab samples that had been previously tested using one of the two RT-qPCR tests was examined using RT-nPCR and the results were compared. It was found that taking both standard RT-qPCR tests together, the RT-nPCR test was able to identify 90% of the detected samples as positive by RT-qPCR. It also detected 13% samples as positive among samples that were negative by the standard RT-qPCR test (likely false negatives). Based on the experimentally measured false negative rate by RT-nPCR tests from this study, it was estimated that as many as 50% of positive samples may escape detection in single pass testing by RT-qPCR in an actual testing scenario.
â€œThis new test is awaiting approval from ICMR. We might ask ICMR to use this test in those places where there are no RT-qPCR machines,â€ remarked Dr Mishra.