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目的基于Allglo探针技术结合多重荧光PCR,建立快速、简便鉴定致病性大肠埃希菌相关毒素基因的方法。方法选取致病性大肠埃希菌的紧密黏附素基因(eae)、志贺样毒素Ⅰ基因(stx I)、志贺样毒素II基因(stx II)和转录激活因子基因(aggR)作为靶点。通过设计引物和Allglo探针,建立多重荧光PCR反应体系。同时,构建了4种毒素基因标准质粒,进而评价方法的特异性、灵敏性和重复性。结果基于Allglo探针技术的多重荧光PCR方法可同时准确、特异地鉴定致病性大肠埃希菌所携带的4种毒素相关基因,eae基因和aggR基因的灵敏度为10 copies/μL,stx I基因和stx II基因的灵敏度为1 copies/μL;定量检测的批间和批内变异系数均小于5%。对356份腹泻粪便样本进行评价,结果显示检出39份基因阳性。39份阳性样本中,eae基因阳性为53.85%,stxⅠ基因和stx II基因阳性为23.08%,aggR基因阳性为46.15%。通过直接测序方法进行鉴定,符合率达到100.00%。结论本实验建立的基于Allglo探针技术结合多重荧光PCR方法具有操作简便、特异性好和灵敏度高等特点,为致病性大肠埃希菌鉴定提供了一种快速、可靠的检测方法。
Objective To establish a rapid and simple method for the identification of pathogenic Escherichia coli-associated toxin genes based on Allglo probe technology combined with multiplex fluorescence PCR. Methods The eae gene, stx I gene, shigetoxin II gene (stx II) and transcriptional activator gene (aggR) of pathogenic Escherichia coli were selected as targets . By designing primers and Allglo probes, a multi-fluorescence PCR reaction system was established. At the same time, four toxin gene plasmids were constructed to evaluate the specificity, sensitivity and repeatability of the method. Results The multiplex PCR method based on Allglo probe technique could simultaneously and accurately identify four toxin-associated genes in pathogenic Escherichia coli. The eae gene and aggR gene had a sensitivity of 10 copies / μL. The stx I gene And the sensitivity of stx II gene was 1 copies / μL; the coefficient of variation of intra-assay and intra-assay were all less than 5%. 356 diarrhea stool samples were evaluated, the results showed that 39 were positive. Among 39 positive samples, the eae gene positive rate was 53.85%, the stx Ⅰ gene level and stx II gene level were 23.08%, and the positive rate of aggR gene was 46.15%. By direct sequencing method for identification, the coincidence rate reached 100.00%. Conclusion Allglo-based probe technology combined with multiplex fluorescence PCR method established in this experiment has the advantages of simple operation, good specificity and high sensitivity, which provides a rapid and reliable method for the identification of pathogenic Escherichia coli.