基于RT-qPCR方法对新冠病毒4种基因型鉴定的可行性研究

Feasibility Study on Identification of Four SARS-CoV-2 Genotypes Based on RT-qPCR

严重急性呼吸道综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的广泛传播强调了对该病毒进行持续监测的紧迫性,以便早发现并采取预防措施。虽然全基因组测序(Whole genome sequencing, WGS)作为SARS-CoV-2检测的金标准,但是其专业性和昂贵成本限制了其在各种情境下的广泛应用。在资源匮乏的地区,迫切需要一种低成本高效率的替代方法。实时荧光定量PCR(Real-timereverse transcription quantitative polymerase chain reaction,RT-qPCR)提供了一种经济、快速的方法,同时有效补充了WGS方法。本研究旨在验证SARS-CoV-2 Omicron BF.7/BA.5.2/XBB/BQ.1及亚分支变异株单重核酸检测试剂盒(RT-qPCR法)的性能,并通过对101例SARS-CoV-2阳性临床样本进行评估,验证其在实际应用中的可行性。研究结果显示,该试剂盒与“野生型”SARS-CoV-2、Alpha、Beta、Delta变异株和甲型流感病毒参考株之间无交叉反应,最低检测限为100拷贝/mL。在101例新冠病毒核酸阳性样本中,该试剂盒对Omicron BF.7/BA.5.2/XBB/BQ.1及亚分支变异的检测结果与WGS检出结果完全一致。因此,该RT-qPCR试剂盒为SARS-CoV-2 Omicron变异株亚型的快速鉴定提供了一种简单、经济且高效的方法,可作为WGS的有效补充方案。

Widespread transmission of severe acute eespiratory ayndrome xoronavirus 2(SARS-CoV-2)underscores the urgency of continuous monitoring of this virus for early detection and preventive measures.Whole genome sequencing(WGS)is the“gold standard”for SARS-CoV-2 detection,but its technical complexity and expense limit its widespread application in various contexts.In resource-limited areas,there is a pressing need for a low-cost and efficient alternative.Real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR)offers an economical and rapid method that complements WGS effectively.We aimed to validate the performance of a RT-qPCR based assay kit for the SARS-CoV-22019-nCoV Omicron subvariants BF.7/BA.5.2/XBB/BQ.1.We also assessed its feasibility in practical application by evaluating 101 clinical samples positive for SARS-CoV-2.The kit did not show cross-reactivity with“wild type”SARS-CoV-2,Alpha,Beta,Delta variants,or influenza A virus reference strains,with a minimum limit of detection of 100 copies/mL.In 101 samples positive for SARS-CoV-2,detection of Omicron BF.7/BA.5.2/XBB/BQ.1 subvariants by the kit was completely consistent with WGS results.Therefore,this RT-qPCR kit provides a simple,economical,and efficient method for rapid identification of SARS-CoV-2 Omicron subtypes,thereby serving as an effective complement to WGS.