论文部分内容阅读
目的建立一种快速检测和鉴定六类致泻性大肠埃希菌和志贺菌的多重PCR方法。方法根据六类致泻性大肠埃希菌和志贺菌的毒力基因eae、stx1、stx2、elt、est Ia、est Ib、aggR、ipa H、afa D及16S rRNA基因rrs建立多重PCR反应体系,优化引物浓度、PCR反应条件等,评价该方法的特异性和灵敏度,并用该方法鉴定本实验室保存的174株致泻性大肠埃希菌和24株志贺菌,将结果与单重PCR、本实验室已建立的鉴定五类致泻性大肠埃希菌和志贺菌的多重PCR方法结果进行对比。结果 10对PCR引物均可特异性扩增相应基因片段。该反应体系对174株致泻性大肠埃希菌和24株志贺菌的鉴定结果与单重PCR检测结果一致,本研究的多重PCR检测下限可达4.56×101CFU/反应(1.14×104CFU/ml)。结论本研究采用毒力基因及内参照基因建立的多重PCR方法可在一个反应体系中鉴定和区分致泻性大肠埃希菌和志贺菌,为临床快速检测、疾病预防控制实验室筛查、腹泻的暴发溯源等提供了方便快捷的技术支持。
Objective To establish a multiplex PCR method for rapid detection and identification of six types of diarrheal Escherichia coli and Shigella. Methods Based on the virulence genes eae, stx1, stx2, elt, est Ia, est lb, aggR, ipa H, afa D and 16S rRNA rrs of six types of diarrheal Escherichia coli and Shigella, PCR-reaction conditions were optimized, the specificity and sensitivity of the method were evaluated, and 174 strains of Escherichia coli and 24 strains of Shigella were identified by this method. The results were compared with single-PCR, The laboratory has established the identification of five types of diarrhea Escherichia coli and Shigella multiplex PCR method for comparison. Results All 10 PCR primers could amplify the corresponding gene fragments specifically. The results of this PCR system were identical with that of single PCR in 174 E. coli isolates and 24 strains of Shigella. The detection limit of multiplex PCR in this study was 4.56 × 101CFU / reaction (1.14 × 104CFU / ml) ). Conclusion The multiplex PCR method established by virulence genes and internal reference genes can identify and distinguish diarrheal Escherichia coli and Shigella in a reaction system. This method is suitable for clinical rapid detection, disease prevention and control laboratory screening, diarrhea The source of such outbreaks provide a convenient and efficient technical support.