大肠杆菌O157∶H7双重荧光PCR方法的建立与应用

摘要:目的　建立快速检测食品中大肠杆菌O157︰H7的双重荧光PCR 方法。方法　针对大肠杆菌O157︰H7菌体抗原O和鞭毛抗原H的特异性基因rfbE（O157）和fliC（H7）筛选设计引物和探针，优化反应条件，建立可特异性检测大肠杆菌O157︰H7的双重荧光PCR方法，并对该法的特异性、敏感性和重复性进行评价，对模拟样品和实际样品进行初步验证。结果　利用建立的方法对2株大肠杆菌O157︰H7及12株非大肠杆菌O157︰H7进行检测，O157︰H7菌株检测结果均为rfbE和fliC阳性，非O157︰H7菌株检测结果均为阴性，rfbE和fliC基因的特异性均为100%；2基因的检测灵敏度可达1.16×102 CFU/ml；批内、批间变异系数均小于3.5％；模拟样品立即检测，2个基因的最低检出限（2.7×102 CFU/ml）和细菌纯培养时接近；实际样品检测结果与我国检验检疫行业标准（SN/T 0973-2010）的检测结果一致。结论　建立的荧光定量PCR 方法特异性及重复性好，灵敏度高，可快速准确鉴定大肠杆菌O157︰H7菌株。

Establishment and application of a dual real-time PCR for the detection of Escherichia coli O157:H7

Abstract: Objective This work was to establish a quick dual real-time PCR method for the detection of Escherichia Coli O157:H7 in food. Methods Specific primers and probes were designed according to Escherichia Coli O157:H7 highly specific gene of rfbE O157 and fliC H7. A dual real-time PCR method was developed by optimizing the reaction conditions and parameters. The specificity, sensitivity and reproducibility of this method were evaluated. The established system was tested by simulated and practical samples. Results PCR method was used to detect 2 strains of E.coli O157:H7 and 12 other E.coli strains. rfbE and fliC were positively detected in Escherichia coli O157:H7 but not detected in non-O157:H7 strains. The specificity of rfbE and fliC were both 100%. The sensitivity of both genes was 1.16×102CFU/ml. Variation coefficients of the intra-assay and the inter-assay over the dynamic range of the TaqMan probe assays were lower than 3.5%. Simulated samples were detected immediately, and the minimum detection limit of rfbE and fliC were both 2.7×102CFU/ml, close to the bacterial pure culture .The results of dual real- time PCR to the practical samples were consistent with those of SN standard. Conclusion The dual real-time PCR is a repeatable, specific, sensitive and rapid method for the detection of EHEC O157: H7.