Abstract
BackgroundReverse transcription-quantitative PCR (RT-qPCR) is commonly used to diagnose SARS-CoV-2, but it has limited sensitivity in detecting the virus in asymptomatic close contacts and convalescent patients. In this study, we propose the use of reverse transcription-digital droplet PCR (RT-ddPCR) to detect SARS-CoV-2 in clinical samples. MethodsThe clinical performance of RT-ddPCR targeting of ORF1ab and N genes was evaluated in parallel with RT-qPCR using 200 respiratory samples collected from close contacts and patients at different phases of infection. ResultsThe limits of detection (LODs) for RT-ddPCR assays were determined using six dilutions of ACCUPLEX SARS-Cov-2 reference material. The LODs of ORF1ab and N genes were 3.7 copies/reaction and 2.2 copies/reaction, respectively. Compared to RT-qPCR, RT-ddPCR increased the positive rate by 12.0% in 142 samples from SARS-CoV-2-infected patients. Additionally, RT-ddPCR detected SARS-CoV-2 in three of 26 specimens from close contacts that tested negative by RT-qPCR, and infection was confirmed using follow-up samples. Finally, RT-ddPCR improved the equivocal results from RT-qPCR in 56.3% (9/16) of convalescent patient samples. ConclusionsDetecting SARS-CoV-2 in samples with low viral loads using RT-qPCR can be challenging. However, our study suggests that RT-ddPCR, with its higher sensitivity and accuracy, is better suited for detecting low viral copies in samples, particularly those from close contacts and convalescent patients.
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