亚欧型及南非型口蹄疫一步法多重荧光rt-pcr检测方法建立_.docVIP

中国农业科学 2010,43(14: Scientia Agricultura Sinica 亚欧型及南非型口蹄疫一步法多重荧光 RT-PCR 检测方法建立 吴绍强,查成刚,邓俊花,林祥梅,刘 建,梅 琳 (中国检验检疫科学研究院,北京 100029 摘要:【目的】口蹄疫是一种严重的“政治经济病”,世界各国面临的口岸疫情防控形势均十分严峻。针对 南非型(SATⅠ,SATⅡ,SATⅢ型潜在入侵中国的严峻态势,为了防止疫情跨境传播,迫切需要建立有效甄别亚欧 型(A、O、C 及亚洲Ⅰ型与南非型口蹄疫的检测方法,以进一步提高防控工作的准确性。【方法】选择口蹄疫 病毒(FMDV基因组保守的 5非翻译区(5UTR基因为靶序列,分别设计亚欧型及南非型 FMDV 特异的荧光 PCR 引物及 Taqman 探针, 通过对反应体系和反应条件的优化, 以插入扩增目的片段的阳性克隆质粒为模板,建立多重 荧光 PCR 检测体系;进一步以体外转录 cRNA 作为阳性质控品,建立亚欧型及南非型 FMDV 一步法多重荧光 RT-PCR 鉴别方法,并进行敏感性、特异性确定及田间样本检测。 【结果】本研究建立的 FMDV 多重荧光 PCR 检测方法,对 亚欧型及南非型 FMDV 阳性质粒的扩增效率均在 90%以上;以倍比稀释的体外转录 cRNA 作为模板,进行敏感性测 试表明,本方法最低可检测至 7.6×10-9ng 南非型 cRNA 和 6.3×10-8ng 亚欧型 cRNA;特异性试验结果显示本方法 可以有效甄别亚欧型与南非型 FMDV;田间样本检测结果与样品实际感染情况一致。【结论】本研究建立的 FMDV 一步法多重荧光 RT-PCR 检测方法敏感、 特异而且快速, 为亚欧型与南非型口蹄疫的鉴别诊断及流行病学调查提供 了新的方法。 关键词:口蹄疫病毒;荧光 RT-PCR;体外转录 cRNA;检测 Identification of Euroasiatic and South Africa Serotype Foot-and-Mouth Disease Virus by One-Step Multiplex Real-Time Quantitative RT-PCR Assay WU Shao-qiang, ZHA Cheng-gang, DENG Jun-hua, LIN Xiang-mei1, LIU Jian, MEI Lin (Chinese Academy of Inspection and Quarantine, Beijing 100029 Abstract:【 Objetive 】 Foot-and-mouth disease (FMD is a serious political and economic disease worldwide and is caused by foot-and-mouth disease virus (FMDV. In order to improve the diagnostic speed and accuracy of FMDV, especially for the early detection of FMDV transmission on the boundary quarantine, the one-step real-time quantitative RT-PCR (RT-qPCR method was established to detect Euroasiatic serotype (EAT and South Africa serotype (SAT FMDV simultaneously. 【 Method 】 Aiming to amplify the conserved 5 untranslated region (UTR of FMDV genomic sequence, the specific primers(U6/L6 and probe(P6 for EAT, primers (AY48U/L and probe (AY48P for SAT of FMDV were designed to establish a multiplex qPCR detection method for FMDV . The amplification solution and reaction conditions were optimized using plasmid inserted with target DNA fragment as template. To offer control for the established qPCR assay, the respe