鸡马立克氏病病毒和疫苗毒株PCR诊断方法的建立及其应用

Establishment and application of PCR diagnosis method for chicken Marek’s disease virus and vaccine virus

研究旨在快速有效地诊断鸡马立克氏病(MD),区分MD血清Ⅰ型病毒(MDV-Ⅰ)与疫苗毒株。试验对Gen Bank发表的140余株MDV序列进行全基因组比对,选定MDV-Ⅰ型特有的meq基因设计1对引物,通过优化退火温度等条件建立了MDV-Ⅰ的PCR诊断方法,评估该方法的特异性、灵敏度及重复性。结果显示,设定退火温度64℃、35个循环时,以MDV-Ⅰ型毒株为模板可扩增得到286 bp的片段;以CVI988或814疫苗毒株为模板时,可扩增得到463 bp的片段;该方法特异性强、灵敏度高,扩增结果稳定,可区分MDV-Ⅰ型毒株和疫苗毒株。应用该方法诊断1例免疫后仍有MD症状的鸡,可同时扩增约286和463 bp的片段;2个片段的测序结果显示,扩增出的片段分别与MDV-Ⅰ型毒株和疫苗毒株相匹配。研究表明,试验建立的PCR方法可快速有效地诊断MDV-Ⅰ型毒株感染鸡并筛选免疫失败的鸡群。

The aim of the study was to rapidly and efficiently diagnose Marek’s disease(MD) in chickens and to distinguish MDV-Ⅰ from vaccine strains. The experiment conducted a genome-wide alignment of more than 140 MDV strains published by Gen Bank, selected MDV-Ⅰ specific meq gene to design a pair of primers, and optimized annealing temperature and other conditions, a PCR diagnosis method for MDV-Ⅰ was established to evaluate the specificity,sensitivity and repeatability of the method. The results showed that when the annealing temperature was 64 ℃and 35 cycles. A 286 bp fragment can be amplified by using MDV-Ⅰ strain as a template, and a 463 bp fragment can be amplified by using CVI988 or 814 vaccine strain as a template. The method has strong specificity, high sensitivity, stable amplification results, and can distinguish MDV-Ⅰ strains from vaccine strains. Using the method to diagnose a chicken with symptoms of Marek’s disease after immunization, the fragments of approximately 286 and 463 bp can be amplified simultaneously. The sequencing results of the two fragments showed that the amplified fragments matched the MDV-Ⅰ strain and the vaccine strain, respectively.The study indicates that the PCR method established in the experiment can quickly and effectively diagnose MDV-Ⅰ strain infection of chickens and screen the flocks of chickens that have failed immunity.