鸡3种主要DNA病毒多重感染复合PCR检测方法的建立

发布时间：2018-01-26 08:31

  本文关键词： 马立克氏病病毒 传染性喉气管炎病毒 鸡痘病毒 DNA病毒 复合PCR　出处：《河南农业科学》2017年07期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:According to the Gen Bank registered chicken Marek's disease virus (MDV), chicken infectious laryngotracheitis virus (ILTV). Primer design for the conserved nucleotide sequence of fowlpox virus (FPVV) genome 3 pairs of primers (9 pairs of primers) were designed for each of the three viruses. The primers were screened by biological software. Three pairs of matching primer pairs were obtained, which were verified by a single PCR. The optimal conditions of PCR reaction were optimized, and the optimal annealing temperature, primer concentration and Taq DNA polymerase concentration were determined. Through specificity, sensitivity test and simplified PCR test, a simple complex PCR detection method was established. The size of the 3 bands amplified by the compound PCR was in accordance with the expected, that is, 228bpnmdv). 400 BP ILTVN 499 BP FPVA; The established composite PCR method can simultaneously detect the DNAs of 100fg MDV / ILTVV FPV, and according to the principle of PCR technology. The classical three temperature thermal cycle is improved to two temperature thermal cycle test, that is, annealing and extension are combined into one step (62 鈩，

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