The study was aimed at establishing a triplex PCR to differentiate M. bovis, M. mycoides subsp. mycoides Small Colony Type and M. agalactiae out of the same sample, for clinical diagnosis and epidemiological survey. Three pairs of primers were designed and synthesized according to the published literatures in GenBank. And a triple PCR was developed to amplify conservative regions of each bacterial genomes of M. Bovis, M. mycoides subsp. mycoides Small Colony Type and M. agalactiae, with the target sequences of 448, 549 and 375 bp, respectively.Sensitivity of this triple PCR was analyzed. Specificity was verified with template from M. gallisepticum, M. Swine, M. agalactiae and M. mycoides subsp. mycoides Small Colony. And reliability was tested by comparing the results from determinative bacteriology. Three specific fragments of 448, 549 and 375 bp, corresponding to M. Bovis, M. mycoides subsp. mycoides Small Colony Type and M. agalactiae, were amplified under optimized condition, without interference between templates and the primers. And results were in agreement with determinative bacteriology. No band was shown in targeting genomes from M. gallisepticum and M. Swine. Sensitivity was determined as 0. 8 ng ·μL-1. These results suggest that the triplex PCR could differentiate M. Bovlis, M. mycoides subsp. mycoides Small Colony Type and M. agalactiae based on one single sample, with high sensitivity, specificity and reliability,and can be applied in clinical diagnosis and epidemiological survey.%本研究旨在建立一种可一次性区分牛支原体、丝状支原体丝状亚种小克隆和无乳支原体的三重PCR诊断方法,为临床诊断和流行病学调查提供可靠检测技术.根据GenBank发表的上述3种病原的基因组序列保守区域设计3对特异性引物建立三重PCR方法;确定其检测敏感性,以猪支原体、鸡支原体、无乳支原体和丝状支原体丝状亚种小克隆基因作模板检验其特异性;同时和病原分离鉴定结果对比其准确性.结果表明在优化体系和条件下能够同时得到扩增长度为448、549、375 bp 3条特异性片段,未扩增出猪、鸡支原体模板特异性片段;其敏感性(可检测到的最小模板DNA含量)为0.8 ng·μL-1;36份临床样品检测结果显示,三重PCR检测结果与分离培养鉴定方法一致,均能鉴定出牛支原体阳性病料.本研究建立的三重PCR诊断方法能够一次性鉴别3种支原体,具有高敏感性、特异性和准确性,可用于临床诊断和流行病学调查.
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