含内参靶标的禽败血支原体和滑液支原体多重荧光PCR检测方法研究  

Multiplex Fluorescence PCR with Internal Amplification Control for Detecting Mycoplasma gallisepticum and Mycoplasma synoviae: Method Design and Validation

作　　者：高志强 汪琳 张红梅 蒲静 赵相鹏 任彤 张伟 赖平安 GAO Zhi-Qiang;WANG Lin;ZHANG Hong-Mei;PU Jing;ZHAOXiang-Peng;REN Tong;ZHANG Wei;LAI Ping-An(China Customs Science and Technology Research Center,Beijing 100026;Beijing Customs,Beijing 100026)

机构地区：[1]中国海关科学技术研究中心,北京100026 [2]北京海关,北京100026

出　　处：《中国口岸科学技术》2022年第11期84-90,共7页China Port Science and Technology

基　　金：国家重点研发计划(2022SWAQ0100);海关总署科研项目(2021HK181)。

摘　　要：在序列比对分析的基础上,分别针对禽败血支原体脂蛋白基因、滑液支原体16S rRNA基因以及β-actin(内参)设计3对引物和3条探针,经体系优化建立了可快速鉴别检测2种禽类最重要的支原体(禽败血支原体和滑液支原体)的多重荧光PCR检测方法。经概率回归分析显示,该方法可分别检出4.48和4.89基因拷贝的DNA,且不与其他禽病病原发生交叉反应。应用建立的荧光PCR检测方法,对送检的358份拭子进行检测,检出败血支原体阳性6份,滑液支原体阳性2份,与OIE推荐的常规PCR方法结果一致。本研究建立的含内参靶标的多重荧光PCR方法不仅特异性强、灵敏度高,而且可对采样和检测全过程进行质控,可用于禽败血支原体和滑液支原体的快速筛查。Based on sequence alignment analysis, three pairs of primers and three probes were designed for the lipoprotein gene of Mycoplasma gallisepticum, 16S rRNA of Mycoplasma synoviae and β-actin(Internal amplification control, IAC). By optimizing the reaction system, a multiplex fluorescence PCR was established for rapid identification and detection of the two most important poultry mycoplasmas(M. gallisepticum and M. synoviae). Probit analysis showed that this method could detect target DNA with 4.48 and 4.89 copies respectively, and had no cross-reaction with other avian pathogens. The established fluorescence PCR detection method was used to detect 358 swabs, with 6 positive samples for M. gallisepticum and 2 positive samples for M. synoviae, which was consistent with results obtained from the conventional PCR method recommended by OIE. The multiplex fluorescence PCR with IAC established in this study was not only specific and sensitive, but also can control the whole process of sampling and detection. It can be used for rapid screening of M. gallisepticum and M. synoviae.

关 键 词：内参靶标 败血支原体 滑液支原体 多重荧光PCR 

分 类 号：S858.3[农业科学—临床兽医学]