免疫捕获PCR法快速检测大肠埃希氏O157∶H7的研究

Abstract

目的建立两种免疫捕获PCR法快速检测E.coliO157∶H7,并探讨其灵敏度和特异性。方法运用免疫磁珠集菌(IMS)和抗体包被扩增管(mACT)两种方法富集待测样品中E.coliO157∶H7,再应用巢式PCR法检测编码E.coliO157∶H7O抗原的rfbE基因,对13株E.coliO157菌和10株非E.coliO157菌进行检测。结果应用两种icPCR检测,所有E.coliO157∶H7和E.coliO157NM菌株均为阳性结果,而其它包括与O157抗血清能交叉凝集的菌株检测结果均为阴性;对于含有48×102cfu/ml以上浓度的E.coliO157∶H7菌悬液,两种方法,所有试验管都能检测到特异性扩增;并证实了这两种方法可用于检测鲜牛奶中E.coliO157∶H7,样品中E.coliO157∶H7的含量只要不少于101cfu/ml,经过6小时增菌均能检测出来。结论IMS-icPCR和mACT-icPCR用于检测E.coliO157∶H7,是快速、灵敏且高特异的方法。

To develop two methods for the rapid detection of E coli 157∶H7 by immunocapture PCR (icPCR) and to investigate their sensitivity and specificity,IMS and mACT were carried out to capture E coli O157∶H7 in test samples and the nest PCR was used to detect the rfbE gene encoding for the production of lipopolysaccharide O side chain of E.coli O157∶H7 in 13 strains of E.coli O157∶H7 and 10 strains of non E.coli O157∶H7. It was found that all the strains of E.coli O157∶H7 and E.coli∶O157∶NM showed positive results as determined by these two methods of immumnocaptured PCR. whereas other strains, including those cross reacting with the O157 antisera,showed negative results.Positive detection of E.coli O157∶H7 was obtainable when the load of organism in the test samples was over 4.8×10 2 cfu/ml.In addition, these two methods could be used for the detection of E.coli O157∶H7 in fresh milk with 6 hours enrrichend,(within 6 hours), when the concentration of this organism was not as few as 10 cfu/ml. It is concluded that IMS icPCR and mACT icPCR appear to be the rapid, sensitive and specific methods for the detection of E.coli O157∶H7 in test samples.