流产衣原体TaqMan实时荧光定量PCR 检测方法的建立及初步应用

Establishment and Preliminary Application of a TaqMan Real-time Fluorescence Quantitative PCR Method for Chlamydia abortus Detection

关键词:  流产衣原体  蛋白酶样活性因子  TaqMan探针  实时荧光定量PCR

Key words:  Chlamydia abortus  Chlamydial protein-like activity factor  TaqMan probe  Real-time Fluorescence Quantitative PCR

摘要:

流产衣原体是导致绵羊地方性流产的主要病原体，给全球畜牧业经济发展构成了巨大威胁。为建立一种灵敏、特异且快速的检测流产衣原体的实时荧光定量PCR方法，依据衣原体蛋白酶样活性因子的基因组序列设计了针对检测流产衣原体的引物和TaqMan探针，对反应体系和反应条件进行了优化，对方法的灵敏度、特异性及重复性进行了评价，并初步应用于临床样本检测。结果显示，该方法的最低检测限为26 copies/μL，灵敏度是普通PCR的10倍；与其他可以引起类似症状的病原体无交叉反应；组内和组间变异系数均小于3%；对156份流产羊拭子的基因组进行检测，检出率为78.21%。研究表明，该方法可以很好的应用于流产衣原体的大规模临床样本检测，为流产衣原体病的高通量检测和流行病学调查提供技术手段。

Abstract:

Chlamydia abortus (C. abortus) is the main pathogen leading to Enzootic abortion of ewes (EAE), which poses a great threat to the economic development of global animal husbandry. To establish a sensitive, specific and rapid real-time fluorescence quantitative PCR method for the detection of C.abortus, primers and TaqMan probe were designed for the detection of C.abortus based on Chlamydial protein-like activity factor (CPAF). The reaction system and reaction conditions were optimized, the sensitivity, specificity and reproducibility of this method were evaluated. The method was preliminarily applied to the detection of clinical samples. The results showed that the detection limit of this method was 26 copies/μL, and the sensitivity was 10 times that of conventional PCR; there was no cross-reaction with other pathogens that could cause similar symptoms; the intra-group and inter-group coefficients of variation were less than 3%. The DNA of 156 aborted sheep swabs were detected with this method, and the detection rate of C.abortus was 78.21%. The results showed that this method could be applied to the detection of large clinical samples of C.abortus, and provided technical method for the high-throughput detection and epidemiological investigation of C.abortus.