鸡3种主要DNA病毒多重感染复合PCR检测方法的建立

依据Gen Bank中注册的鸡马立克氏病病毒(MDV)、鸡传染性喉气管炎病毒(ILTV)、鸡痘病毒(FPV)基因组核苷酸序列中的保守序列设计引物,3种病毒各设计3对引物,共计9对引物,经生物学软件筛选后,得到3对相互匹配较佳的引物。3对匹配引物经单一PCR验证后,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聚合酶浓度;经特异性、敏感性试验及简化PCR试验,建立简易复合PCR检测方法。复合PCR扩增出的3条带大小与预期一致,即228 bp(MDV)、400 bp(ILTV)、499 bp(FPV);建立的复合PCR方法能同时检测出100 fg MDV、ILTV、FPV的DNA。同时依据PCR技术原理,将经典的三温热循环改进为二温热循环试验,即将退火和延伸合并为一步(62℃),缩短了反应时间。建立的3种病毒的复合PCR检测方法具有较高的特异性和敏感性,可以用于临床病料的快速诊断。

Establishment of Multiplex PCR Detection Method for Three Kinds of Main DNA Viruses in Chicken

Specific primers were designed upon the conservative sequences of Marek's disease virus (MDV),infectious laryngotracheitis virus (ILTV),and fowlpox virus (FPV) genomic nucleotide sequences from registered in GenBank.Three pairs of specific primers for each virus were designed,a total of 9 pairs of primers for three viruses(MDV,ILTV,FPV) were screened by biological software,and three pairs of primers matched each other.After verification of three pair suitable primers with singleplex PCR,the reaction conditions of the multiplex PCR were optimized,including annealing temperature,concentrations of primers and Taq DNA polymerase.The simple multiplex PCR assay was successfully established after specificity,sensitivity and simplified test.The specific bands with lengths of 228 bp(MDV),400 bp (ILTV),499 bp(FPV) were amplified by the multiplex PCR,which were consistent with those expected.The results demonstrated that the method could simultaneously amplify these three viruses at a sensitivity of 100 fg when all three viruses were present.According to the principle of PCR technology,the classical PCR(a three step thermal cycle assay) was improved to be a two step thermal cycle assay,namely annealing and extension step were merged into one step (62 ℃),and the reaction time was shortened.This multiplex PCR for three DNA viruses was both highly specific and sensitive,and could be used as a rapid diagnostic assay for clinical samples.