畜牧兽医学报2017，Vol.48Issue(8)：1491-1498,8.DOI:10.11843/j.issn.0366-6964.2017.08.013
猪肺炎支原体和猪鼻支原体双重荧光定量PCR检测方法的建立
Duplex Real-time PCR Method for Detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis
摘要
Abstract
The study was conducted to develop a TaqMan probe-based,sensitive,specific duplex real-time PCR for detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.The specific primers and probes,labeled with FAM and Texas Red respectively,were designed to amplify the P97 gene of Mycoplasma hyopneumoniae and P37 gene of Mycoplasma hyorhinis.The reaction system of duplex real-time PCR was established and optimized,and its sensitivity,specificity and repeatability were analyzed.The sensitivity of duplex real-time PCR established was 10 copies · μL-1 for Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.There was no cross reaction with other common viral and bacterial pathogens.The concentration of standard coefficient of variation of Ct values was less than 5％,indicating good reproducibility.Moreover,72 clinical samples were detected by the duplex real time PCR assay.The results showed that the positive rates of Mycoplasma hyopneumoniae were 80％,22％ and 58.33％ in lung tissues,nasal swabs and bronchoalveolar lavage fluids samples,respectively;the positive rates of Mycoplasma hyorhinis were 40％,66％ and 41.67％ in lung tissues,nasal swabs and bronchoalveolar lavage fluids samples,respectively.The duplex real-time PCR for detecting Mycoplasma hyopneumoniae and Mycoplasma hyorhinis simultaneously was more sensitive than the common PCR,and can be used for detecting clinical samples.The advantage of this method is that it can acquire various information of samples in a short time,and save the time and economic consumption of multiple tests,and it provides a new and reliable detection technology for detection and control of Mycoplasma h yopneumoniae and Mycoplasma hyorhinis.关键词
猪肺炎支原体/猪鼻支原体/双重荧光定量PCRKey words
Mycoplasma hyopneumoniae/Mycoplasma hyorhinis/duplex real-time PCR分类
农业科技引用本文复制引用
武昱孜,熊祺琰,刘蓓蓓,张珍珍,王佳,华利忠,韦艳娜,邵国青..猪肺炎支原体和猪鼻支原体双重荧光定量PCR检测方法的建立[J].畜牧兽医学报,2017,48(8):1491-1498,8.基金项目
江苏省农业科技自主创新资金项目[CX(14)2040] （14）
肉类生产与加工质量安全控制协同创新中心 （）
