鸡4种主要RNA病毒病多重感染复合PCR检测方法的建立  

Establishment of Multiplex PCR Detection Method for Four Kinds of Avian Main RNA Viral Diseases

作　　者：刘媛[1] 李学伍[2] 王丽[2] 谷雨[3] 王林建[3] 

机构地区：[1]河南科技大学动物科技学院,河南洛阳471023 [2]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [3]河南农业大学生物工程学院,河南郑州450002

出　　处：《河南农业科学》2016年第11期105-109,共5页Journal of Henan Agricultural Sciences

基　　金：公益性行业(农业)科研专项(201303033)

摘　　要：依据Gen Bank中注册的禽流感病毒(AIV)、鸡传染性支气管炎病毒(IBV)、新城疫病毒(NDV)和传染性法氏囊病毒(IBDV)基因组核苷酸序列,通过序列比对获得AIV、IBV、NDV、IBDV的相对保守序列。根据每种病毒的保守序列利用生物学软件分别设计3对特异性引物,4种病毒共设计12对引物,并对12对引物进行模拟筛选,获得匹配最佳的4对引物。对获得的最佳匹配引物进行单项PCR验证,优化复合PCR反应条件,确定最佳的退火温度、引物浓度和Taq DNA聚合酶使用量;经特异性、敏感性及简化PCR试验,最终建立了简易复合PCR检测方法。结果表明,建立的检测方法可同时扩增出4条大小与设计相符的特异性片段,即IBV(146 bp)、NDV(215 bp)、IBDV(345 bp)、AIV(435 bp);建立的复合PCR方法具有较强的敏感性和特异性,能检测出100 pg AIV、IBV、IBDV的cDNA和100 fgNDV的cDNA;将三温热循环改进为二温热循环,即将退火和延伸过程合为一步(62℃),大大缩短了反应时间。Specific primers were designed upon gene conservative sequence in GenBank as well as the gene of avian influenza virus （AIY） , avian infectious bronchitis virus （IBY） ,newcastle disease virus （NDY） and infectious bursal disease virus（IBDY） genomic nucleotide. Biological software were uesd to design 3 pairs of specific primers in the conserved sequence of each kind of virus, 12 pairs of primers were designed for 4 viruses, 12 pairs of primers was simulated screened, and 4 pairs of primers were appropriate. Optimal annealing temperature, concentration of primers and Taq DNA polymerase concentration were determined to optimizated multiplex PCR reaction conditions. The specificity test, sensitivity test and simplified PCR test were used to establish the simple multiplex PCR detection method. The results showed that specific bands at lengths of IBY （ 146 bp） , NDY （215 bp） , IBDY （345 bp） and AIY （435 bp） respectively were amplified by the developed quadruple PCR, which were consistent with those expected. It indicated that high specificity and sensitivity of the developed method,which could detect 100 pg AIY,IBY,NDY IBDY cDNA and 100 fg NDY cDNA. According to the classical principle of PCR technology, the composite PCR （three step thermal cycle step） was improved, which anneal ing and extension step were merged into one step,and the optimum temperature of anneal ing-extension was 62 ℃, which could reduce the reaction time.

关 键 词：禽流感病毒 鸡传染性支气管炎病毒 新城疫病毒 传染性法氏囊病毒 RNA病毒 复合PCR 

分 类 号：S855.3[农业科学—临床兽医学]