Designing of rapid assay for the detection of RdRp/Orf1ab specific to SARS-CoV-2

SARS-CoV-2 is still threat and mostly used detection method is real time reverse transcriptase polymerase chain reaction (rRT-PCR) for the open reading frame (Orf1ab), RNA-dependent RNA polymerase (RdRp), nucleocapsid (N) and envelope (E) genes of virus. However, rRT-PCR may have false negative rate for the nucleic acid detection. Since the RdRp/Orf1ab has high sensitivity for the molecular detection, two sandwich models, Model 1A-Model 1B, based on hybridization on lateral flow assay (LFA) were designed here and applied with the synthetic and clinical samples of RdRp/Orf1ab. In this purpose colloidal gold nanoparticles (AuNPs) were used as label. Membranes having different flow rate, three oligonucleotide probe concentrations and running buffers were used. Although synthetic target sequence was recognized by all the LFAs, PCR products obtained from either the synthetic plasmid DNA or oro/nasopharyngeal swabs were detected by Model 1 A using W12 membrane. Designed strip assays detected the RdRp/Orf1ab of the clinical samples as 100% sensitivity and specifity. It means that they might be used for the detection of virus and can be modified for the recognition of mutant genes of virus. These findings also demonstrated the importance of membranes, sandwich models, probe concentrations and sample contents for developing LFAs for viral detection.