中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2012年
6期
401-404
,共4页
周为民%陆柔剑%耿合员%李辉%段希洁%杨扬%谭文杰
週為民%陸柔劍%耿閤員%李輝%段希潔%楊颺%譚文傑
주위민%륙유검%경합원%리휘%단희길%양양%담문걸
冠状病毒属%分子诊断技术%核酸类
冠狀病毒屬%分子診斷技術%覈痠類
관상병독속%분자진단기술%핵산류
Coronavirus%Molecular diagnostic techniques%Nucleic acids
目的 建立2012年确定的新冠状病毒的分子检测方法,并筛选优化引物与探针.方法 依据最先发表的208 bp长的新冠状病毒1b片段核酸序列,比对分析后设计合成常规RT-PCR引物1对及荧光定量RT-PCR引物1对与TaqMan探针2条(TZ1,TZ2),同时合成锁核酸修饰的TaqMan探针2条(LNA-TZ1,LNA-TZ2).建立检测新冠状病毒感染的常规RT-PCR方法与4种荧光定量RT-PCR,分析比较其灵敏度与特异性,同时参照欧洲发表的2种荧光定量RT-PCR引物与探针对(upE,ORF1b)建立相应方法,以合成的阳性模板比较6对荧光定量RT-PCR引物与探针的检出灵敏度.结果 所合成的常规RT-PCR与荧光定量RT-PCR引物与探针皆有较好特异性,不能扩增其他人冠状病毒模板及常见呼吸道病毒模板,检出下限达50 ～ 500拷贝/反应.锁核酸修饰TaqMan探针可改善荧光定量PCR检测方法的反应性能,经锁核酸修饰的TaqMan探针与常规TaqMan探针相比,检出率提高10倍左右,其中LNA-TZ1与upE探针对具最佳反应性能.结论 本研究所建立的常规RT-PCR与荧光定量RT-PCR可用于新冠状病毒的分子检测,并推荐使用LNA-TZ1与upE探针对.本研究为新冠状病毒分子检测方法应用与改进奠定了基础.
目的 建立2012年確定的新冠狀病毒的分子檢測方法,併篩選優化引物與探針.方法 依據最先髮錶的208 bp長的新冠狀病毒1b片段覈痠序列,比對分析後設計閤成常規RT-PCR引物1對及熒光定量RT-PCR引物1對與TaqMan探針2條(TZ1,TZ2),同時閤成鎖覈痠脩飾的TaqMan探針2條(LNA-TZ1,LNA-TZ2).建立檢測新冠狀病毒感染的常規RT-PCR方法與4種熒光定量RT-PCR,分析比較其靈敏度與特異性,同時參照歐洲髮錶的2種熒光定量RT-PCR引物與探針對(upE,ORF1b)建立相應方法,以閤成的暘性模闆比較6對熒光定量RT-PCR引物與探針的檢齣靈敏度.結果 所閤成的常規RT-PCR與熒光定量RT-PCR引物與探針皆有較好特異性,不能擴增其他人冠狀病毒模闆及常見呼吸道病毒模闆,檢齣下限達50 ～ 500拷貝/反應.鎖覈痠脩飾TaqMan探針可改善熒光定量PCR檢測方法的反應性能,經鎖覈痠脩飾的TaqMan探針與常規TaqMan探針相比,檢齣率提高10倍左右,其中LNA-TZ1與upE探針對具最佳反應性能.結論 本研究所建立的常規RT-PCR與熒光定量RT-PCR可用于新冠狀病毒的分子檢測,併推薦使用LNA-TZ1與upE探針對.本研究為新冠狀病毒分子檢測方法應用與改進奠定瞭基礎.
목적 건립2012년학정적신관상병독적분자검측방법,병사선우화인물여탐침.방법 의거최선발표적208 bp장적신관상병독1b편단핵산서렬,비대분석후설계합성상규RT-PCR인물1대급형광정량RT-PCR인물1대여TaqMan탐침2조(TZ1,TZ2),동시합성쇄핵산수식적TaqMan탐침2조(LNA-TZ1,LNA-TZ2).건립검측신관상병독감염적상규RT-PCR방법여4충형광정량RT-PCR,분석비교기령민도여특이성,동시삼조구주발표적2충형광정량RT-PCR인물여탐침대(upE,ORF1b)건립상응방법,이합성적양성모판비교6대형광정량RT-PCR인물여탐침적검출령민도.결과 소합성적상규RT-PCR여형광정량RT-PCR인물여탐침개유교호특이성,불능확증기타인관상병독모판급상견호흡도병독모판,검출하한체50 ～ 500고패/반응.쇄핵산수식TaqMan탐침가개선형광정량PCR검측방법적반응성능,경쇄핵산수식적TaqMan탐침여상규TaqMan탐침상비,검출솔제고10배좌우,기중LNA-TZ1여upE탐침대구최가반응성능.결론 본연구소건립적상규RT-PCR여형광정량RT-PCR가용우신관상병독적분자검측,병추천사용LNA-TZ1여upE탐침대.본연구위신관상병독분자검측방법응용여개진전정료기출.
Objective To develop and optimize the molecular detection assays for recently identified human coronavirus (HCoV) infection.Methods Based on the 208 base pair(bp) sequence of novel HCoV reported by HPA of UK,we designed and obtained several pairs of primer(F-1,R-1 ;F-2,R-2) and Taqman probes(TZ1,TZ2) for detection of novel HCoV.Two of probes were modified with LNA(LNA-TZ1,LNA-TZ2).Then,RT-PCR and various real time RT-PCR assays were developed and optimized in this study.We also compared our assays with the real time RT-PCR assays reported recently by Europe team based on upE or ORF1b target.Results The RT-PCR or real time RT-PCR assays for novel HCoV were developed without cross-reactivity with other HCoV and several common respiratory viruses using clinical specimen panel.The analytical sensitivity of assays were less than 50-500 copies per reaction and the detection was improved when Taqman probe modified with LNA-tagged,compared to no LNA-tagged in real time RT-PCR assays.The upE and LNA-TZ1 based assays were better than others.Conclusion The molecular detection sensitivity and specificity of TaqMan-based real time PCR assay could be improved when probe tagged with LNA.The upE or LNA-TZ1 based real time RT-PCR assay was recommend for detection of novel HCoV.This study laid a foundation for improving the performance of novel HCoV detection.
