鸡马立克氏病强弱病毒株PCR鉴别检测方法在诊断中的应用评价

Assessments of a PCR method for detection and identification of virulent Marek's disease virus and vaccine strain for Marek's disease diagnosis

为在现地准确诊断鸡马立克氏病(MD)以及区分发病鸡中MD病毒(MDV)野毒株和疫苗株的感染,本研究选择MDV血清1型(MDV-1)基因组中两个特有的、强弱毒株存在序列差异的基因片段132 bp重复序列(132 bpr)和meq基因作为靶基因,建立了MDV的PCR检测方法。特异性试验和敏感性试验表明该PCR方法特异性强,敏感性高,132 bpr和meq基因两对引物对阳性重组质粒的检测下限分别为1.0×103拷贝/μL和1.0×102拷贝/μL。通过对近10年的1057份临床样品的检测对该PCR方法进行应用评价。结果表明病毒分离与PCR检测结果的符合率为100%,PCR检测结果可信度高;琼脂扩散试验(AGP)对MDV PCR检测阳性样品的检测阳性率为86.25%,PCR法检测敏感性明显高于AGP法。此外,对分离病毒株meq基因序列分析结果表明,所有分离株均具有中国野毒株特征,即突变主要出现于其编码蛋白的6个氨基酸中(K77E、D80 Y、V115 A、T139 A、P176 R或P217A),表明该PCR检测方法具有良好的特异性。本研究建立的MDV PCR检测方法特异性强、灵敏度高,具有良好的实用性,可以用于MD的临床诊断。

To rapidly diagnose Marek＇s disease （MD） in chickens and distinguish virulent Marek＇s disease virus （MDV） from vaccine strain in poultry farms, a PCR assay was developed for detecting MDV with 2 pairs of primers targeting 132 bp repeat sequence and meq gene of MDV, which had different size in virulent MDVs （132 bpxl to 132 bpx3 for 132 bp repeat sequences and 1,020 bp for meq gene） and serotype I MDV vaccine strain （multiple copies for 132bp repeat sequence and 1,197 bp or 1,200 bp for meq gene）. The results of specificity test and sensitivity test showed that the PCR assay showed high specificity and sensitivity, and the detection limits of two primers were 1.0 ×10^3 copies/μL and 1.0 ×10^2 copies/μL in positive recombinant plasmids, respectively. We evaluated the PCR assay by detection of 1,057 clinical samples accumulated during the last decade. The results of virus isolation were 100% consistent with the PCR assay. Meanwhile, the PCR showed higher sensitivity than that of agar gel precipitation （AGP）, where positive rate of AGP was only 86.25%. Moreover, the analysis of meq sequences from isolates showed that the deduced amino acid sequences contained the characteristic point mutations in the MDVs prevalence in China, mainly occurred among K77E, D80Y, V115A, T139A, P176R or P217A. The results suggested that the PCR assay could be applied for the detection field MDVs and diagnose MD in chickens.