一步法多重RT-PCR禽流感病毒分型诊断法的建立与应用  

Establishment and application of single-step multiplex RT-PCR for avian Influenza virus type and subtype detection

作　　者：吕长荣[1,2] 乔海莲[1] 杨增岐[1] 陈杰[3] 顾节清[1,2] 李晓成[3] 

机构地区：[1]西北农林科技大学动物科技学院 [2]农业部动物检疫所国家动物流行病学研究中心,山东青岛266032 [3]农业部动物检疫所国家动物流行病学研究中心

出　　处：《西北农林科技大学学报（自然科学版）》2007年第4期63-67,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家科技攻关项目(2002BA514A-15-3)

摘　　要：根据禽流感病毒(AIV)的M基因序列设计合成了1对AIVM基因的通用诊断引物AMDI／AMD2,针对H5N1、H9N2两种亚型的HA基因分型设计合成了2对分型诊断引物H15D1／H5D2,H9D1／H9D2。采用一步法多重RT-PcR技术建立了一种禽流感病毒快速分型诊断方法,并对其特异性和灵敏度进行验证。结果表明,该诊断方法速度快,一般只需要28～30 h即可鉴定出结果,比病毒分离鉴定(5～7 d)至少缩短4～6 d;特异性强,试验组53株AIV全部扩增出与预期同样长度的目的条带,与病毒分离及血清学鉴定方法的检测结果完全一致。对照组中新城疫病毒(NDV)、减蛋综合征病毒(EDS76V)、传染性支气管炎病毒(IBV)均未扩增出特异性基因条带,;灵敏度高,试验组将AIV接种SPF鸡胚,培养后的鸡胚尿囊液(AAF)进行10-2稀释后,仍可以检测到其中的AIV。The pair of primer AMD1 and AMD2 were designed based on the strongly conservative region of the avian influenza virus（AIV） M gene,which hold specific property of A type AIV. The different subtype diagnosis primers H5D1/H5D2, and HgD1/HgD2 were respectively designed based on the HA gene of H5N1 and HgN2 subtype AIV. The single-step RT-PCR was established with three pairs of primers. The method of specificity and sensitivity were verified. The results showed that the specific electropho- resis stripes were 380 bp length for M gene,431 bp length for HSA gene and 387 bp length for HgA gene by RT-PCR with the three sets of primers. The PCR products showed the same length with the expected one. But the controlled NDV,EDS76V,IBV were not amplified by the RT-PCR. The shortest time of diagnosis was 28 hours,which was much shorter than other AIV diagnosis methods. The AIV could also be detected after those allantoic-amniotic fluid （AAF） were diluted 100 times. Fifty-three AIV and fifty samples were detected successfully,which were completely consistent with the results tested by serum methods. So the AIV detection via RT-PCR is very specific,sensitive,safe and time-saving. Thus this RT-PCR method is suited for application of AIV diagnosis in common laboratories.

关 键 词：禽流感病毒 M基因 HA基因 RT-PCR 诊断方法 

分 类 号：S852.659.5[农业科学—基础兽医学]