Objectives

The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primer/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection.

Methods

We compared the clinical performances of the recommended primer/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID‐19. An additional 30 “pre-intervention screening” samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets.

Results

The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives.

Conclusion

An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.

中文翻译：

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如何为 SARS-CoV-2 基因组检测选择合适的实时 RT-PCR 引物组？

目标

SARS-CoV-2 大流行引发了对快速大规模诊断检测的前所未有的需求，以促进临床和公共卫生干预。目前，临床和公共卫生实验室正在使用世界卫生组织推荐的几种定量逆转录聚合酶链反应 (RT-qPCR) 检测方法，通常针对 RNA 依赖性 RNA 聚合酶 (RdRp)、包膜 (E) 区域和核衣壳 (N) 编码区。但是，目前尚不清楚不同测试的结果是否具有可比性。本研究旨在阐明由美国疾病预防控制中心和 Charité/Berlin 设计的引物/探针组的临床性能，以帮助临床实验室选择 SARS-CoV-2 常规检测的检测方法。

方法

我们使用来自临床诊断为 COVID-19 的患者的一百个鼻咽拭子标本比较了推荐引物/探针组的临床性能。本研究还包括来自未怀疑患有 COVID-19 的患者的另外 30 个“干预前筛查”样本。我们还在 31064 个欧洲 SARS-CoV-2 和相关基因组变体与推荐的引物/探针组之间进行了序列比对。

结果

本研究表明，世界卫生组织推荐的引物/探针组在 SARS-CoV-2 RNA 检测灵敏度方面存在显着差异，尤其是对于低水平病毒载量。数千个 SARS-CoV-2 序列的比对表明，引物/探针结合位点的遗传多样性仍然相对较低。然而，多个核苷酸错配可能会导致假阴性。

结论

了解目标基因和引物/探针组的局限性可能会影响临床实验室对分子检测分析的选择。