Rapid visual detection of SARS-CoV-2 by colorimetric loop-mediated isothermal amplification

Evaluation of the performance of a new set of primers defined from the ORF1ab sequence, and its combination with a previously published set of primers from the N sequence, to detect SARS-CoV-2 RNA by the loop-mediated isothermal amplification technique is presented. The ORF1ab primer set enables visual detection of SARS-CoV-2 RNA in 16 min. In addition, a simultaneous reaction with both ORF1ab and N primers allows for higher sensitivity of detection, particularly when low numbers of copies are present (250 viral RNA copies). Further, the protocol is able to detect viral RNA in saliva samples. The procedure reported could be easily implemented in the generation of a new and sensitive rapid point-of care device for SARS-CoV-2 RNA visual detection. METHOD SUMMARY Optimized loop-mediated isothermal amplification reactions were carried out using the combination of two sets of primers (designed on ORF1ab and N sequences). Saliva samples were pretreated by heating and proteinase K, before SARS-CoV-2 determination by loop-mediated isothermal amplification.

Automated summary

The study evaluated the performance of a new set of primers defined from the ORF1ab sequence and its combination with a previously published set of primers from the N sequence for detecting SARS-CoV-2 RNA using loop-mediated isothermal amplification technique. The results showed that the ORF1ab primer set enabled visual detection of SARS-CoV-2 RNA in 16 minutes, and a simultaneous reaction with both ORF1ab and N primers allowed for higher sensitivity of detection, particularly when low numbers of copies were present (250 viral RNA copies). Additionally, the protocol was able to detect viral RNA in saliva samples, demonstrating the potential for developing a new and sensitive rapid point-of-care device for visual detection of SARS-CoV-2 RNA.