One-Stop Extraction and In Situ RT-qPCR for Ultrasensitive Detection of Highly Diluted SARS-CoV-2 in Large-Volume Samples from Aquatic Environments.

Surveillance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in aquatic environments attracted attention due to its considerable impacts on human health and ecology, especially in countries with poor sanitation standards. Based on a strategy of one-stop extraction and in situ amplification, we developed an ultrasensitive method that uses a polyacrylamide derivative-modified filter disc (PAD-FD), in which highly diluted RNA can be efficiently concentrated onto the filter disc and directly used for amplification. A newly designed spin column with a cup-like filter base facilitated the non-contact transfer of the affinity filter disc from the column to a PCR tube. The limit of detection of the PAD-FD coupled with RT-qPCR is 10 copies/mL. Using 32 suspected SARS-CoV-2 samples, we demonstrated that the detection rate of our method (62.5%, 20/32) was triple the rate of the commercial kit (18.8%, 6/32). Using a PAD-FD, 56.3% (18/32) and 40.6% (13/32) of the 10-fold-dilution samples with river and tap water, respectively, were detected. Even when diluted 100-fold, 28.1% (9/32) and 37.5% (12/32) were still detected in river and tap water, respectively. We believe that the PAD-FD method offers an accurate testing tool for monitoring viral RNA in aquatic environments, contributing to the forewarning of the SARS-CoV-2 outbreak and the breaking of the transmission chain.

Simultaneous detection and differentiation of SARS-CoV-2, influenza A virus and influenza B virus by one-step quadruplex real-time RT-PCR in patients with clinical manifestations.

OBJECTIVE: To develop a novel quadruplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, differential diagnosis and detection of co-infections. METHODS: A one-step quadruplex rRT-PCR assay was developed for simultaneous detection and differentiation of SARS-CoV-2 ORF1ab and N genes, influenza A virus (hIAV) and influenza B virus (hIBV). RESULTS: The quadruplex rRT-PCR assay had good sensitivity and specificity. Correlation coefficients and amplification efficiencies of all singleplex and quadruplex rRT-PCR reactions were within acceptable ranges. The 95% lower limits of detection for plasmid standards and positive nucleic acid extracts of the quadruplex rRT-PCR assay were 57.38-95.11 copies/µL and 114.65-154.25 copies/µL, respectively. Excellent results were attained for inter- and intra-assay reproducibility. Among these clinical samples, only four samples showed results inconsistent with the singleplex rRT-PCR assays. CONCLUSIONS: To the authors' knowledge, this is the first study to report a quadruplex rRT-PCR assay for the detection of two SARS-CoV-2 genes, hIAV and hIBV with perfect clinical performance.