RT-qPCR is the current gold standard for detecting viral RNA including SARS-CoV2. It is a two-step assay involving the reverse transcription of the RNA followed by amplification of cDNA and requires 60 mins per sample. To reduce the assay time, a novel isothermal approach using exponential amplification reaction (EXPAR) and an uncommon reverse transcription free (RTF) conversion of RNA to DNA is introduced. EXPAR amplifies DNA at a single temperature, thus avoiding lengthy heating and cooling steps found in PCR. RTF-EXPAR is a ‘one-pot’ detection assay and can accurately identify 7.25 copies per μL of SARS-CoV2 RNA in less than 10 minutes.
RTF-EXPAR involves the binding of a 30-mer DNA oligonucleotide known as binder DNA to the SARS-CoV2 RNA. This leads to the formation of RNA/DNA heteroduplex which is cleaved using the restriction enzyme BstN1 to generate a short trigger DNA which is then rapidly amplified using exponential amplification reaction (EXPAR). These trigger DNA sequences are shorter than those in PCR and LAMP and are less likely to undergo sample degradation. The amplified strand is then detected using fluorescence. Upon comparing this approach with PCR and LAMP it was found that, the speed of this amplification reaction was faster, with 7.25 copies per μL of SARS-CoV2 RNA detected in under 10 min and 1,450 copies per μL detected under 35 mins, a significant improvement over both RT-qPCR and RT-LAMP.
Thus, the RTF-EXPAR technique for the detection of SARS-CoV-2 is advantageous when compared to the other existing methods for its speed and easy handling.
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