同时检测六种人类冠状病毒的单管多重RT-PCR方法的建立

A novel one-tube multiplex RT-PCR assay for simultaneous detection of six human coronaviruses

目的 建立一种单管多重RT-PCR检测方法,同时检测6种人类冠状病毒.方法 由GenBank获取6种人类冠状病毒（HCoV-NL63、HCoV-229E、SARS-CoV、HCoV-OC43、HCoV-HKU1和MERS-CoV）的核苷酸序列作为参比,分别设计6种冠状病毒保守区靶序列特异性引物,用本实验室所保存的病毒株及核酸样本为模板建立基于全自动电泳仪分析的单管多重RT-PCR检测技术,进行了检测限和重复性评价,并用本实验室所保存的其他呼吸道病毒阳性样品对该方法的特异性进行了再验证.用140份临床样本结合荧光定量RT-PCR法平行验证了该检测方法的可行性.结果 基于全自动电泳仪分析的多重RT-PCR检测技术可同时检测6种人类冠状病毒,阳性标本均显示至少一条相应特异性产物预期大小片段（分别为195、304、332、378、415、442 bp）,阴性对照无特异性条带显示,具有较高的特异性.在检测单个病毒时检测限能达到1.0×10^1～ 1.0×10^2拷贝/μl.其他呼吸道病毒验证未发现交叉反应.140份临床样本平行验证结果与常用的单一HCoV检测的荧光定量RT-PCR法一致,阳性样本均为28份（20％）.结论 建立的基于全自动电泳分析的单管多重PCR方法能够同时检测6种人类冠状病毒感染,有较高的灵敏度和特异性.

Objective To develop an one-tube multiplex RT-PCR assay for simultaneous detection of six human coronaviruses（HCoVs）.Methods Genbank sequences of six human coronaviruses,including HCoV-NL63,HCoV-229E,SARS-CoV,HCoV-OC43,MERS-CoV,and HCoV-HKU1,were included as reference sequences.Primers were designed based on multiple alignment of reference sequences,targeting the conserved regions of each species of HcoV.Virus strains and nucleic acid preserved in our lab were used as template in developing this automatic-electrophoresis-based one-tube multiplex RT-PCR assay.Detection limits and reproducibility were also evaluated with these templates.Samples with infection of other respiratory viruses preserved in our lab were used to evaluate specificity of this assay.Finally,we tested this assay with 140 clinical samples that were validated by real-time PCR in parallel.Results This automatic-electrophoresis-based multiplex RT-PCR assay was able to detect six human coronaviruses simultaneously.All positive samples in this study were detected with at least one specific fragment of anticipated length （195,304,332,378,415,442 bp）.No fragment was detected in negative controls.Detection limits of 1.0 × 10^1-1.0 × 10^2 copies/μ1 were achieved in tests of single virus.No cross reaction was observed with other respiratory viruses.This multiplex RT-PCR assay showed the same sensitivity and specificity to that of individual real-time RT-PCR validated with clinical samples.Both methods detected 28 positive samples （20%）.Conclusions Six HCoVs can be detected in one tube by this novel multiplex RT-PCR assay with high sensitivity and specificity.