禽流感病毒、新城疫病毒、猪瘟病毒和口蹄疫病毒多联RT-PCR诊断技术的建立  

Establishment of Multiple RT-PCR Diagnostic Techniques for Avian influenza virus (AIV), Newcastle disease virus (NDV), Classical swine fever virus (CSFV) and Food and mouth disease virus (FMDV)

作　　者：刘维全[1] 刘海鹏[2] 江禹[3] 王本旭[3] 王吉贵[1] 杨淑艳[3] 孙绍光[3] 涂长春[3] 刘芃芃[1] 

机构地区：[1]中国农业大学生物学院,北京100094 [2]沈阳农业大学畜牧兽医学院,沈阳110161 [3]解放军军需大学军事兽医研究所,长春130062

出　　处：《农业生物技术学报》2005年第1期92-95,共4页Journal of Agricultural Biotechnology

摘　　要：选择禽流感病毒(AIV)、新城疫病毒(NDV)、猪瘟病毒(CSFV)和口蹄疫病毒(FMDV)基因组序列高保守区,按照多联PCR引物的设计要求,利用DNAsis计算机软件设计并合成了4对特异性扩增引物,扩增片段大小分别为470、320、200和140bp。以AIV、NDV、CSFV和FMDV的培养物提取RNA并反转录,进行RT-PCR特异性扩增。结果表明,各单项RT-PCR扩增产物与设计的4对引物之间的序列大小一致。在分别建立了各病毒单项RT-PCR及AIV与NDV、CSFV与FMDV二联RT-PCR的基础上,进一步优化各种多联PCR扩增条件,成功建立了上述4种病毒的四联RT-PCR技术。经特异性和灵敏度实验证明,本方法具有高度的特异性和灵敏性,其灵敏度为10pg总RNA。在3h内即可完成样品的检测。The homology of the sequences, reported and registered in GenBank of different strains of Avian influenza virus(AIV), Newcastle disease virus(NDV), Classical swine fever virus(CSFV)and Food and mouth disease virus(FMDV), was respectively analyzed and compared with each other. According to the properties of these viruses, the conservative domain of M gene for AIV, F gene for NDV, 5′non-coding domain end for CSFV and 2B gene for FMDV were selected for PCR amplification. In order to prevent the formation of conformational dimers between different primers, four pairs of primers designed with the DNAsis system under the condition of G+C(50%~60%), 18~25 bp in length and Tm (72~85), were analyzed with VNTI5.5 system. The specific fragments amplified were as follows: 141 bp for FMDV, 200 bp for CSFV, 319 bp for NDV and 471 bp for AIV. The optimal conditions of PCR for each virus mentioned above were determined by orthogonal assay, and 2 or 4 of the 4 pairs of primers were then combined and used for amplification trials. The results showed that four specific fragments in different length would be successfully amplified in one tube at the same time. The products of PCR were tested to be specific by sequencing. Of 46 pathological samples detected with the multiple PCR, five were AIV, seven were NDV, fifteen were CSFV and six were FMDV. The amplifications above were identified by single PCR. Meanwhile, the results were corresponded to those of electronic microscopy, HA and ELISA assay. The method described here was very practicable and sensitive, specific, simple and cheap. It could be used in diagnosis of AIV, NDV, CSFV and FMDV for different animals.

关 键 词：CSFV FMDV 口蹄疫病毒 猪瘟病毒 AIV 禽流感病毒 新城疫病毒 RT-PCR 特异性 扩增 

分 类 号：S852[农业科学—基础兽医学] S858[农业科学—兽医学]