复合探针实时荧光PCR检测大肠杆菌O157:H7

DETECTION OF EHEC O157:H7 BY USING COMPLEX PROBE REAL-TIME FLUORESCENT PCR

目的建立一种快速、准确、特异的肠出血性大肠杆菌O157:H7的定量检测方法。方法以rfbE基因作为待检靶基因,根据复合探针荧光定量分析原理设计检测引物和探针,建立检测大肠杆菌O157:H7的实时荧光定量PCR方法。并对本方法检测的特异性、灵敏度、重复性等进行评价。结果所有O157:H7的检测结果均为阳性,而所有其它菌株检测结果均为阴性;该方法检测的灵敏度可达10 cfu.mL-1,定量检测的批间和批内差异均小于5%。在模拟污染牛奶中,可检出1×101cfu.μL-1的细菌。结论本文建立的复合探针实时荧光定量PCR技术能快速准确、特异、敏感地对大肠杆菌O157:H7进行定量分析,为大肠杆菌O157:H7的检测提供了新的方法。

Object To develop a method for rapid, sensitive and specific detection for Escherichia coli （EHEC） O157： H7. Method Based on the principle of complex probe and fluorescent quantitative analysis, a real - time PCR assay targeted at rfbE was developed. Result It showed good specificity, sensitivity and repeatability. All 5 strains of EHEC O157 ： H7 tested were positively detected and none of other enteric bacteria was detected by the assay. The sensitivity of the assay was about 10 cfu·mL^-1 in pure cultures. The coefficient of variation of intra - assay and inter - assay was less than 5 %. The ability of this assay to detect food samples was assessed with artificially contaminated milk. The seeded bacterial concentration of 10 cfu·μL^-1 can be detected by the assay in artificially contaminated milk. Conclusion The real- time PCR assay is a reproducible, specific and sensitive method for the detection of EHEC O157：H7.