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

发布时间：2018-01-27 15:26

本文关键词： 猪肺炎支原体猪鼻支原体双重荧光定量PCR　出处：《畜牧兽医学报》2017年08期 　论文类型：期刊论文

【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10copies·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]:A dual fluorescent quantitative PCR method was established for the simultaneous detection of mycoplasma pneumoniae and mycoplasma suis. The specific primers of P97 sequence and P37 sequence of Mycoplasma suis were used. Two TaqMan probes were labeled with FAM and Texas Red, respectively, to establish and optimize the double real-time fluorescence quantitative PCR reaction conditions and systems, and to detect their sensitivity. Specificity and repeatability. The minimum detection value of double fluorescence quantitative PCR for Mycoplasma suis and Mycoplasma suis was 10 copies 路渭 L -1; There was no cross reaction with porcine pathogenic bacteria, viruses and other common bacteria. The coefficient of variation of Ct value of each concentration standard was less than 5 and the reproducibility was good. Among 72 clinical samples, the positive rate of mycoplasma pneumoniae in lung was 80 and the positive rate of nasal swab was 22%. The positive rate of bronchoalveolar lavage fluid was 58.33; The lung positive rate of Mycoplasma suis was 40%, the positive rate of nasal swab was 66g and the positive rate of bronchoalveolar lavage fluid was 41.67.The established double fluorescent quantitative PCR method was more sensitive than that of ordinary PCR. This method can be used to detect mycoplasma pneumoniae and mycoplasma suis rapidly and quantitatively at the same time and obtain multiple information of the sample in a short time. It can quickly solve the time and economic consumption of multiple detection, and provide a new and reliable detection technology for the surveillance and control of Mycoplasma pneumoniae and Mycoplasma suis.
【作者单位】：江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心;省部共建国家重点实验室培育基地-江苏省食品质量安全重点实验室;
【基金】：江苏省农业科技自主创新资金项目[CX(14)2040] 肉类生产与加工质量安全控制协同创新中心
【分类号】：S858.28
【正文快照】： 猪支原体肺炎(Mycoplasmal pneumonia of swine)的主要病原是猪肺炎支原体(Mycoplasma hyopneumoniae,M.hyopneumoniae),是一种慢性、高传染性、可接触性的动物呼吸道疾病[1-2],发病率高、死亡率低,在世界各地的猪场广泛流行和传播[3-6],引起的猪呼吸道疾病综合征(porcine res

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