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
5期
384-387
,共4页
房师松%李娟%王昕%刘涛%程小雯%吕星%吴春利%郑青%张仁利%程锦泉
房師鬆%李娟%王昕%劉濤%程小雯%呂星%吳春利%鄭青%張仁利%程錦泉
방사송%리연%왕흔%류도%정소문%려성%오춘리%정청%장인리%정금천
流感病毒,B型%聚合酶链反应%荧光抗体技术
流感病毒,B型%聚閤酶鏈反應%熒光抗體技術
류감병독,B형%취합매련반응%형광항체기술
Influenza B virus%Polymerase chain reaction%Flourescent antibody tecnique
目的 建立一种新型的双重荧光PCR诊断方法,用于B型流感病毒By (B/Yamagata)和Bv(B/Victoria)亚系的准确分子分型.方法 从GenBank随机下载By和By HA(hemagglutinin)基因各50条序列,通过MEGA分析,利用Primer Primer软件设计亚系特异性引物和通用探针,建立双重荧光PCR诊断方法.用HAI(hemagglutination inhibition)实验确认的B型流感病毒亚系分离毒株和A型流感病毒进行特异性验证,用体外转录核酸拷贝数进行灵敏度实验.结果 2006-2010流感监测年份,对17 765份流感样病例咽拭标本中分离到B型流感病毒793株,本方法鉴定有152株By和641株Bv病毒,与HAI鉴定结果一致.本诊断方法的检测特异性达100％,灵敏度达102拷贝/μl,重复性变异系数＜3.5％.结论 本研究所建立的荧光PCR方法为流感实时监测提供了有力的技术支撑,适合于流感监测实验室对流感病毒的快速分子诊断.
目的 建立一種新型的雙重熒光PCR診斷方法,用于B型流感病毒By (B/Yamagata)和Bv(B/Victoria)亞繫的準確分子分型.方法 從GenBank隨機下載By和By HA(hemagglutinin)基因各50條序列,通過MEGA分析,利用Primer Primer軟件設計亞繫特異性引物和通用探針,建立雙重熒光PCR診斷方法.用HAI(hemagglutination inhibition)實驗確認的B型流感病毒亞繫分離毒株和A型流感病毒進行特異性驗證,用體外轉錄覈痠拷貝數進行靈敏度實驗.結果 2006-2010流感鑑測年份,對17 765份流感樣病例嚥拭標本中分離到B型流感病毒793株,本方法鑒定有152株By和641株Bv病毒,與HAI鑒定結果一緻.本診斷方法的檢測特異性達100％,靈敏度達102拷貝/μl,重複性變異繫數＜3.5％.結論 本研究所建立的熒光PCR方法為流感實時鑑測提供瞭有力的技術支撐,適閤于流感鑑測實驗室對流感病毒的快速分子診斷.
목적 건립일충신형적쌍중형광PCR진단방법,용우B형류감병독By (B/Yamagata)화Bv(B/Victoria)아계적준학분자분형.방법 종GenBank수궤하재By화By HA(hemagglutinin)기인각50조서렬,통과MEGA분석,이용Primer Primer연건설계아계특이성인물화통용탐침,건립쌍중형광PCR진단방법.용HAI(hemagglutination inhibition)실험학인적B형류감병독아계분리독주화A형류감병독진행특이성험증,용체외전록핵산고패수진행령민도실험.결과 2006-2010류감감측년빈,대17 765빈류감양병례인식표본중분리도B형류감병독793주,본방법감정유152주By화641주Bv병독,여HAI감정결과일치.본진단방법적검측특이성체100％,령민도체102고패/μl,중복성변이계수＜3.5％.결론 본연구소건립적형광PCR방법위류감실시감측제공료유력적기술지탱,괄합우류감감측실험실대류감병독적쾌속분자진단.
Objective To develop a novel sensitive duplex real-time PCR assay for accurately identifying B/Yamagata and B/Victoria lineages of influenza virus type B.Methods 50 HA (hemagglutinin) gene sequences coding for B/Yamagata and B/Victoria lineage,respectively,were randomly downloaded for GenBank and analyzed by software MEGA.Primers and probes specific for HA gene of B/Yamagata and B/Victoria lineages were designed by Primer Primer and then applied in the duplex real-time RT-PCR method that was followed developed.Influenza virus B type and A type isolated in our laboratory and typing-confirmed by HAI method were used as reference strains to determine the specificity of this assay and the sensitivity of the duplex amplification was evaluated by viral load testing in terms of in vitro transcribed RNA copy number.Results In 2006-2010,793 influenza virus type B strains were isolated from 17 765 throat swab samples,among which 152 strains were differentiated as By lineage and 641 as Bv lineage by this assay.These results was agreement with that determined by HAI assay.This developed assay allows to accurately identify approximately 102 copies/μl for Bv and By lineage virus with intra-and intercoefficient of variation (CV) ＜ 3.5％ and nearly 100％ specificity.Conclusions This method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination of influenza virus,which could be applied in influenza surveillance laboratories for rapid molecular diagnosis.
