一种用于检测唾液中 SARS-CoV-2 的多重、配对池液滴数字 PCR 检测方法。

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva.

机构信息

Prion Research Center, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, USA.

Colorado State University, Fort Collins, CO, 80523, USA.

出版信息

Sci Rep. 2023 Feb 22;13(1):3075. doi: 10.1038/s41598-023-29858-5.

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-h turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

针对 SARS-CoV-2 大流行，我们开发了一种多重、配对池滴式数字 PCR（MP4）筛选检测方法。该检测方法的关键特点是使用经最少处理的唾液样本、8 个样本配对池以及针对 SARS-CoV-2 核衣壳基因的逆转录滴式数字 PCR（RT-ddPCR）。检测限分别为单个样本和混合样本中每微升 2 个和 12 个拷贝。使用 MP4 检测方法，我们每天可常规处理 1000 多个样本，周转时间为 24 小时，在 17 个月的时间内，共筛查了超过 25 万份唾液样本。模型研究表明，随着病毒流行率的增加，8 个样本池的效率降低，而通过使用 4 个样本池可以减轻这种影响。我们还提出了一种策略，并提供了支持数据，用于创建第三个配对池，作为在高病毒流行率下采用的额外策略。