IMSA技术快速检测肠出血大肠杆菌O157:H7方法的建立及应用

Development and Application of IMSA for Rapid Detection of Enterohaemorrhage Escherichia coli O157:H7

为了建立和评价一种适合食药监及基层检验部门使用的肠出血性大肠杆菌O157:H7快速检测方法,针对肠出血性大肠杆菌O157:H7特异性rfbE基因,应用等温多自配引发扩增技术(Isothermal Multiple Self-matching-initiated Amplification,IMSA),设计引物进行检测。对建立的方法进行特异性试验和灵敏度试验,与荧光定量PCR法进行比对;利用建立的IMSA法确定人工污染样本被检出的最短增菌时间和最低检测灵敏度;使用IMSA法和国标法对24份不同肉制品进行检验,评估两者的一致性。结果表明:该方法仅对肠出血性大肠杆菌O157:H7特异性扩增;IMSA法的灵敏度比qPCR法高,达8.9×10^(2) CFU/mL;人工污染菌种样本最短增菌10 h可以被建立的IMSA法检出,检出限为9.1 CFU/25 g;24份肉制品样本的IMSA法与常规培养法结果一致性为100%。结论:IMSA技术检测肠出血性大肠杆菌O157:H7具有特异性强、灵敏度高、结果快速准确的特点,可在11 h内完成食品中O157:H7的检出,适用于食药监及基层检验部门进行肠出血性大肠杆菌O157:H7的快速检测。

To establish and evaluate a rapid detection method for EHEC O157:H7 suitable for food and drug administration and grassroots inspection departments.To study the specific rfbE gene of EHEC O157:H7,using Isothermal multiple self-matching-initiated amplification(IMSA)to design and perform testing.The specificity test and sensitivity test were carried out and compared with the fluorescence quantitative PCR method.The IMSA method was used to determine the shortest incubation time and the lowest detection sensitivity of artificial contaminated samples.IMSA method and national standard method were used to examine 24 different meat products to evaluate their consistency.The results showed that:This method only amplified EHEC O157:H7 specifically.The sensitivity of IMSA was higher than that of qPCR,up to 8.9×10^(2) CFU/mL.The IMSA method could be used to detect artificially contaminated bacterial samples for the shortest time of 10 h,and the detection limit was 9.1 CFU/25 g.The IMSA method was 100%consistent with the conventional culture method in 24 meat samples.Conclusion:IMSA technology has the characteristics of strong specificity,high sensitivity,rapid and accurate resμLts,and can be used to detect EHEC O157:H7 in food within 11 hours.It is suitable for food and drug administration and basic inspection departments to detect EHEC O157:H7 quickly.