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
2010年
5期
383-385
,共3页
杨亮%张晓梅%张晓光%马晶%王敏%温乐英%王大燕%白天%舒跃龙%钱永华%曾毅
楊亮%張曉梅%張曉光%馬晶%王敏%溫樂英%王大燕%白天%舒躍龍%錢永華%曾毅
양량%장효매%장효광%마정%왕민%온악영%왕대연%백천%서약룡%전영화%증의
聚合酶链反应%杂交%流感
聚閤酶鏈反應%雜交%流感
취합매련반응%잡교%류감
Polyomerase chain reaction%Hybridization%Influenza virus
目的 建立一种基于多重RT-PCR结合反向斑点杂交技术的多种流感病毒核酸快速检测方法.方法 对5种亚型流感病毒的HA基因序列进行比对分析,选择合适的保守区域设计引物序列,并在扩增产物序列内部选择高变区设计探针序列.首先,使用生物素标记的引物扩增出同样带标记的PCR产物;然后,产物与膜上的探针杂交,通过生物素-亲和素反应,最后以斑点显色的方式来检测和鉴别不同亚型的流感病毒.实验中还通过摸索不同的引物浓度和探针浓度来优化反应条件.结果 成功建立了一种基于多重RT-PCR结合反向斑点杂交技术的检测方法,该方法能够成功检测和鉴别5种不同亚型的流感病毒,检测灵敏度比传统的RT-PCR方法高100～1000倍.结论 成功建立了一种可高通量快速检测多种流感病毒的多重RT-PCR结合反向斑点杂交技术的检测方法.
目的 建立一種基于多重RT-PCR結閤反嚮斑點雜交技術的多種流感病毒覈痠快速檢測方法.方法 對5種亞型流感病毒的HA基因序列進行比對分析,選擇閤適的保守區域設計引物序列,併在擴增產物序列內部選擇高變區設計探針序列.首先,使用生物素標記的引物擴增齣同樣帶標記的PCR產物;然後,產物與膜上的探針雜交,通過生物素-親和素反應,最後以斑點顯色的方式來檢測和鑒彆不同亞型的流感病毒.實驗中還通過摸索不同的引物濃度和探針濃度來優化反應條件.結果 成功建立瞭一種基于多重RT-PCR結閤反嚮斑點雜交技術的檢測方法,該方法能夠成功檢測和鑒彆5種不同亞型的流感病毒,檢測靈敏度比傳統的RT-PCR方法高100～1000倍.結論 成功建立瞭一種可高通量快速檢測多種流感病毒的多重RT-PCR結閤反嚮斑點雜交技術的檢測方法.
목적 건립일충기우다중RT-PCR결합반향반점잡교기술적다충류감병독핵산쾌속검측방법.방법 대5충아형류감병독적HA기인서렬진행비대분석,선택합괄적보수구역설계인물서렬,병재확증산물서렬내부선택고변구설계탐침서렬.수선,사용생물소표기적인물확증출동양대표기적PCR산물;연후,산물여막상적탐침잡교,통과생물소-친화소반응,최후이반점현색적방식래검측화감별불동아형적류감병독.실험중환통과모색불동적인물농도화탐침농도래우화반응조건.결과 성공건립료일충기우다중RT-PCR결합반향반점잡교기술적검측방법,해방법능구성공검측화감별5충불동아형적류감병독,검측령민도비전통적RT-PCR방법고100～1000배.결론 성공건립료일충가고통량쾌속검측다충류감병독적다중RT-PCR결합반향반점잡교기술적검측방법.
Objective To establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses. Methods Obtain the HA nucleotide sequences of seasonal influenza H1 N1, seasonal influenza H3N2, influenza H1 N1 and human avian influenza H5 N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance. Results Successfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity.Conclusion Successfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.
