CAJ | 학술논문

为建立检测禽流感病毒（AIV ）且同时区分 H3N2亚型AIV 的方法，本研究根据 H3、N2亚型AIV HA、NA基因及AIV最保守M 基因的保守区域，设计并筛选出3对特异性引物，通过优化反应条件，建立了AIV H3N2亚型和M 基因三重RT‐PCR的检测方法。对该法进行特异性及敏感性检测，并通过该三重RT‐PCR方法对96份临床样品进行检测。结果显示，H3N2亚型AIV可扩增出3条特异性条带，其中518 bp为AIV M 基因、418 bp为N2亚型AIV NA基因、271 bp为H3亚型AIV HA基因；H3亚型和N2亚型AIV均可扩增出2条特异性条带，大小分别为518 bp、271 bp和518 bp、418 bp；其他亚型AIV可扩增出一条特异性条带，大小为518 bp；常见禽病病原体均未扩增出任何条带。敏感性试验表明该法对 H3N2亚型AIV的检测下限为100 pg ；96份临床样品检测结果与病毒分离鉴定结果一致。本研究所建立的AIV H3N2亚型和M 基因三重RT‐PCR为一种简便、快速、有效的检测方法。
위건립검측금류감병독（AIV ）차동시구분 H3N2아형AIV 적방법，본연구근거 H3、N2아형AIV HA、NA기인급AIV최보수M 기인적보수구역，설계병사선출3대특이성인물，통과우화반응조건，건립료AIV H3N2아형화M 기인삼중RT‐PCR적검측방법。대해법진행특이성급민감성검측，병통과해삼중RT‐PCR방법대96빈림상양품진행검측。결과현시，H3N2아형AIV가확증출3조특이성조대，기중518 bp위AIV M 기인、418 bp위N2아형AIV NA기인、271 bp위H3아형AIV HA기인；H3아형화N2아형AIV균가확증출2조특이성조대，대소분별위518 bp、271 bp화518 bp、418 bp；기타아형AIV가확증출일조특이성조대，대소위518 bp；상견금병병원체균미확증출임하조대。민감성시험표명해법대 H3N2아형AIV적검측하한위100 pg ；96빈림상양품검측결과여병독분리감정결과일치。본연구소건립적AIV H3N2아형화M 기인삼중RT‐PCR위일충간편、쾌속、유효적검측방법。
In order to establish a method to detect avian influenza virus (AIV ) and separating H3N2 sub‐types ,three pair of specific primer were designed and selected according to the conserved sequences of AIV matix(M)gene、the hemagglutinin (HA) genes of H3 subtype AIV and the Neuraminidase(NA) genes of N2 subtype AIV ,the reaction conditions were optimized and established the multiplex RT‐PCR .To fur‐ther evaluate the reliability ,the specificity and sensitivity of assay were evaluated ,and 96 clinical samples were detected by the multiplex RT‐PCR .The results show that the H3N2 subtype AIV could be amplified three specific bands by this multiplex PCR .The lengths of these bands were 518 bp (M ) ,418 bp (N2 AIV) and 271 bp (H3 AIV) .The H3 and N2 subtype AIV could be amplified two specific bands ,which were 518 bp and 271 bp as well as 518 bp and 418 bp ,respectively .Other subtypes AIV couble be ampli‐fied a 518 bp specific band .However ,no specific band was amplified from other avain pathogenic virus . The detection limit of the multiplex PCR was 100 pg of H3N2 subtype AIV ;the detection of clinical sam‐ples were accorded with the viral isolation completely .This study provide a simple、quick and effective method for the detection of AIV and separation of H 3N2 subtype AIV .