Establishment of Nucleic Acid Detection Methods for Large-scale Screening of Viruses
Objective To develop rapid and simple nucleic acid assays for large-scale screening of epidemic viruses.Methods The droplet digital polymerase chain reaction(ddPCR)system,which integrates droplet generation,cyclic amplification,and signal read-ing,was used to amplify nucleic acids and monitor the fluorescence signal of each droplet targeting the ORF1ab and N genes of severe a-cute respiratory syndrome coronavirus 2(SARS-CoV-2)and human glyceraldehyde-3-phosphate dehydrogenase(GAPDH)gene as internal reference gene.Results The integrated DDPCR instrument operated on the simple principle of"sample in-result out."Its wa-ter-oil system was compatible with the SARS-CoV-2 assay kit's reagents,and the droplets were stable and unfused by the end of am-plification,allowing real-time fluorescence detection to distinguish different fluorescence signals in a large number of droplets.Droplets with triple fluorescence signals(FAM,HEX,CY5)were observed as early as the 9th minute under thermostatic amplification conditions.By fitting,the real-time fluorescence signal during amplification resembled the real-time polymerase chain reaction amplification curve,including the baseline,exponential,and plateau phases.The amplification curves of several hundred droplets of each gene showed that there was no specific amplification in each droplet,while the results of Ct value analysis showed that the amplification signal could be ob-tained at the 12th minute at the earliest for identification purposes.Conclusion The integrated ddPCR instrument is simple to operate and reacts quickly in droplets,allowing for rapid detection.Thermostatic amplification requires less reaction equipment and is suitable for large-scale batch reactions,whereas photo-type signal acquisition can record the fluorescence signal of a large number of droplets for large-scale screening.
Integrated droplet digital polymerase chain reactionSARS-CoV-2Thermostatic amplification
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