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

doi:10.11843/j.issn.0366-6964.2017.08.013

摘要/Abstract

摘要：

建立同时检测猪肺炎支原体和猪鼻支原体的双重荧光定量PCR方法。根据猪肺炎支原体P97序列和猪鼻支原体P37序列的特异性引物，分别标记FAM和Texas Red荧光报告基团的2条TaqMan探针，建立和优化双重实时荧光定量PCR反应条件和体系，同时检测其敏感性、特异性和重复性。建立的双重荧光定量PCR对猪肺炎支原体和猪鼻支原体的最低检测值均为10 copies·μL^-1；与猪源致病菌、病毒和其他常见细菌均无交叉反应；各浓度标准品的Ct值变异系数小于5%，重复性好。检测72份临床样本，其中猪肺炎支原体的肺阳性率为80%，鼻拭子阳性率22%，支气管肺泡灌洗液阳性率58.33%；猪鼻支原体的肺阳性率为40%，鼻拭子阳性率66%，支气管肺泡灌洗液阳性率41.67%。建立的双重荧光定量PCR方法比普通PCR更加敏感，实用性强，可用于临床样品的检测。该方法能同时快速和定量地检测猪肺炎支原体和猪鼻支原体，在短时间内获得样品的多个信息，快速地解决了多次检测所需的时间以及经济消耗，为猪肺炎支原体和猪鼻支原体的监测和防控提供了新型、可靠的检测技术。

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 hyopneumoniae and Mycoplasma hyorhinis.

中图分类号:

S858.28

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.

WU Yu-zi, XIONG Qi-yan, LIU Bei-bei, ZHANG Zhen-zhen, WANG Jia, HUA Li-zhong, WEI Yan-na, SHAO Guo-qing. Duplex Real-time PCR Method for Detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(8): 1491-1498.

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