鸡毒支原体SYBR Green I荧光定量PCR检测方法的建立  

Development of a SYBR Green I real-time PCR assay for detection of Mycoplasma gallisepticum

作　　者：孙俊颖[1,2,3] 刘志成[1,2,3] 孙敏华[1,2,3] 李冰玲[4] 沈海燕[1,2,3] 张春红[1,2,3] 董嘉文 李林林[1,2,3] 张建峰[1,2,3] 

机构地区：[1]广东省农业科学院动物卫生研究所,广东广州510640 [2]广东省畜禽疫病防治研究重点实验室,广东广州510640 [3]广东省兽医公共卫生公共实验室,广东广州510640 [4]北京出入境检验检疫局,北京100026

出　　处：《中国预防兽医学报》2015年第12期938-942,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国际科技合作项目(2014DFA31730;2012B050500013;2013J4500031);国家自然科学基金(31302101);广东省科技计划项目(2014A040401048)

摘　　要：为建立鸡毒支原体(MG)的快速定量检测方法,本研究根据GenBank中登录的MG 16S rDNA基因序列设计了一对引物,以MG基因组DNA为模板扩增其16S rDNA基因片段,并克隆于pMD18-T载体中,以纯化的重组质粒作为标准品建立了MG SYBR GreenⅠ荧光定量PCR检测方法。结果表明,该方法在1.96×10~8拷贝/μL^1.96×10~2拷贝/μL范围内线性关系良好,相关系数为0.999;最低检测限可达1.96×10~1拷贝/μL;与禽类其它呼吸道病原无交叉反应,特异性良好;批内及批间变异系数均低于2%,具有较高的重复性和稳定性。采用该方法抽检30份疑似临床样品,检出率为40%,敏感性明显高于常规PCR(33%)和血清平板凝集试验方法(23%),检测活疫苗可达到精确定量每头份2.0×10~7拷贝/μL。该研究为MG的早期诊断与净化、疫苗质量监测提供了一种新的快速定量检测技术。To develop a specific assay for quantitative detection of Mycoplasma gallisepticum, a SYBR Green I real-time PCR assay was established with a pair of primers targeting M.gallisepticum 16S rDNA gene. A recombinant plasmid of pMD-16S containing 186 bp of 16S rDNA target fragment was constructed as a standard template to set up the standard curve which exhibited a dynamic linear ranging from 1.96× 108 copies/μL to 1.96×102 copies/μL. Then, the specific and sensitive tests showed that real-time PCR assay was only detected M.gallisepticum with the detection limit of 1.96 ×10-1 copies/μL, but had no cross-reaction with other avian respiratory tract pathogens examined. The variations of both inter- and intra-assay were less than 2%. Moreover, thirty clinical samples were examined for M.gallisepticum, the positive rate was 40% by real-time PCR, but it was only 33% and 23% with conventional PCR and serum plate agglutination test, respectively, showing that this assay was much moresensitive than conventional detection assays. The results showed that this real-time PCR assay provides an effective method for quantitative detection and vaccine quality control of M.gallisepticum.

关 键 词：鸡毒支原体 SYBR Green I 荧光定量PCR 

分 类 号：S852.62[农业科学—基础兽医学]