Quantitative analysis of trace SARS-CoV-2 nucleic acid on cold-chain food and packaging material surfaces using multi chip-based digital PCR

ObjectiveTo address the false-negative risk associated with trace contamination of severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2） on cold-chain food and packaging material surfaces， this study aimed to develop a highly sensitive and quantifiable multiplex detection method.MethodBased on multi chip-based digital PCR （multi cdPCR） technology， we simultaneously targeted the ORF1ab， N， and E genes of SARS-CoV-2. Several parameters were optimized， including primer concentration， probe， and annealing temperature， and the methodological performance was evaluated. Additionally， the viral nucleic acid load was analyzed under simulated contamination conditions on cold-chain food and packaging material surfaces.ResultsThe multi cdPCR detection method could simultaneously identify all three gene fragments of SARS-CoV-2. The detection limit for the ORF1ab gene was 1.36 copies/μL， while the detection limits for the N and E genes are both 0.96 copies/μL. This method demonstrated high specificity， accuracy， and good repeatability， with coefficient of variation less than 1.7%. Analysis of 70 blinded， presumptively positive samples showed positive rates of 72.9% for multi cdPCR， whereas real-time fluorescence quantitative PCR gave a rate of 11.4%. Moreover， the decline of viral nucleic acid load with increasing temperature was more pronounced on food surfaces than on packaging material surfaces.ConclusionThe multi cdPCR method established in this study enables the detection of low viral load of SARS-CoV-2 on cold-chain food and packaging material surfaces， thereby offering a practical tool for the safety supervision of cold-chain foods.