CAJ | 학술논문

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1.0×101～ 1.0×102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.
목적 건립일충단관다중RT-PCR검측방법,동시검측6충인류관상병독.방법 유GenBank획취6충인류관상병독(HCoV-NL63、HCoV-229E、SARS-CoV、HCoV-OC43、HCoV-HKU1화MERS-CoV)적핵감산서렬작위삼비,분별설계6충관상병독보수구파서렬특이성인물,용본실험실소보존적병독주급핵산양본위모판건립기우전자동전영의분석적단관다중RT-PCR검측기술,진행료검측한화중복성평개,병용본실험실소보존적기타호흡도병독양성양품대해방법적특이성진행료재험증.용140빈림상양본결합형광정량RT-PCR법평행험증료해검측방법적가행성.결과 기우전자동전영의분석적다중RT-PCR검측기술가동시검측6충인류관상병독,양성표본균현시지소일조상응특이성산물예기대소편단(분별위195、304、332、378、415、442 bp),음성대조무특이성조대현시,구유교고적특이성.재검측단개병독시검측한능체도1.0×101～ 1.0×102고패/μl.기타호흡도병독험증미발현교차반응.140빈림상양본평행험증결과여상용적단일HCoV검측적형광정량RT-PCR법일치,양성양본균위28빈(20％).결론 건립적기우전자동전영분석적단관다중PCR방법능구동시검측6충인류관상병독감염,유교고적령민도화특이성.
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 × 101-1.0 × 102 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.