猴小肠结肠炎耶尔森氏菌实时荧光PCR快速检测方法的建立

Establishment of Real-time Fluorescent PCR for Rapid Detecting Yersinia Enterocolitica in Monkey

目的 建立一种能快速检测猴小肠结肠炎耶尔森氏菌实时荧光PCR方法.方法 以小肠结肠炎耶尔森氏菌耐热肠毒素基因的保守区为靶区域设计特异引物和探针,建立一种能快速检测小肠结肠炎耶尔森氏菌的实时荧光PCR方法,并对方法的特异性、敏感性、线性相关性、重复性等因素进行试验.结果 建立的荧光PCR法对小肠结肠炎耶尔森氏菌具有特异的反应性,最低检出限为3.3×10 cfu/ml,菌液浓度在3.3×10〈＇7〉cfu/ml至3.3×10cfu/ml时与Ct值有较好的线性关系,试验重复性良好.结论 建立的小肠结肠炎耶尔森氏菌实时荧光PCR检测方法适用于猴小肠结肠炎耶尔森氏菌的快速检测.

Objective To rapidly detect the Yersinia enterocolitica in monkey ,a real time fluorescent PCR assay was established. Method The primers and probe were designed based on the conserved sequence of heat-labile enterotoxin gene of Yersinia enterocolitica. Then PCR reaction system was optimized and evaluated. Results The established real-time PCR method was sensitive and specific to 3.3×10 cfu/ml bacteria concentration. There was reliable correlation between the template copies （3.3 ×10^1 cfu/ml-3.3×10^7cfu/ml） and the Ct value. Conclusion The real-time PCR assay was proved to be specific with high sensitivity and efficiency, and would be a powerful diagnosis method of Yersinia enterocolitica in monkey.