High sensitivity detection of SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method

Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrate its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produces clean characteristic target peaks of defined sizes, which allows for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel and tested a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.

目前已有多种检测方法被应用或报道于严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的诊断与监测工作中。其中，逆转录聚合酶链反应（reverse transcription polymerase chain reaction，RT-PCR）是当前灵敏度最高的检测手段，据称可检出约5个病毒拷贝。但据文献报道，仅47%~59%的阳性病例可通过RT-PCR得以识别，这可能源于采样过程中病毒RNA的丢失或降解，甚至病毒基因组发生突变。因此，开发高灵敏度的检测方法对于保障可靠的检测能力而言至关重要。为提升检测灵敏度并适配各类应用场景，本研究开发了一种基于多重聚合酶链反应（multiplex-PCR）的检测方法，该方法包含172对特异性引物，并验证了其在低病毒拷贝数下检出SARS-CoV-2的效能。该检测体系可产生条带尺寸明确的清晰特征性靶标峰，可通过电泳直接完成阳性样本的鉴定。此外，配套的可选测序步骤既可进一步确认检测结果，还可获取检出病毒的系统发育信息以实现特定毒株的鉴别，这对于作为全球公共卫生核心要务的病毒监测工作而言具有极高的重要性。最后，本研究还同步开发并测试了一种基于多重PCR的宏基因组检测方法，该方法可检出SARS-CoV-2，其额外优势在于能够在低测序深度下揭示病毒的突变多样性及潜在的新型病原体。