用最低检出限评价大肠埃希菌O157:H7的检测方法及应用研究

Evaluation of Escherichia coli O157: H7 Detection Method by the Lowest Detection Limit and Its Application

目的用最低检出限评价大肠埃希菌O157:H7的三种检测方法,在突发公共卫生事件及食源性致病菌流行病学调查中,将大肠埃希菌O157:H7的检测方法有机组合,提高检测的速度、灵敏度。方法将标准菌株10倍稀释,制备模拟样品,用大肠埃希菌O157:H7的三种方法进行检测,比较不同检测方法的最低检出限。结果标准菌株10倍稀释改良CHROMagar O157显色琼脂平板分离法、免疫磁珠分离法、实时荧光PCR法检测法的最低检出限分别为103、100、102cfu/ml;模拟样品中增菌前改良CHROMagar O157显色琼脂平板分离法、免疫磁珠分离法、实时荧光PCR法检测法的最低检出限分别为105、101、104cfu/ml,增菌培养后改良CHROMagar O157显色琼脂平板分离法、免疫磁珠分离法、实时荧光PCR法检测法的最低检出限分别为102、100、100cfu/ml。结论免疫磁珠分离法在增菌前和增菌培养后的检出限为最低,增菌培养后实时荧光PCR法的检出限也达到了最低,适用于食物中毒及食源性疾病的快速诊断。用改良CHROMagar O157显色琼脂平板分离法进行检测,易造成漏检。将免疫磁珠分离法、实时荧光PCR法有机组合,可以大大提高检测的速度,灵敏度,并且可获得菌株。

Objective To evaluate three Escherichia coli O157： H7 detection methods by the lowest detection limit,so as to realize organic combination of the methods in public health emergencies and foodborne pathogen epidemiological surveys and improve the detection rate and sensitivity.Methods Simulation samples were prepared by diluting the standard strain of Escherichia coli O157：H7 10 times and were tested by three Escherichia coli O157：H7 detection methods.The lowest detection limits（LDLs） of the three methods were compared.Results For the standard strains in 10 times dilution,the LDLs of modified CHROMagar O157 agar plate separation（MCO157APS）,magnetic activated cell sorting（MACS） and real-time fluorescence PCR（RTF-PCR） were 103 cfu/ml,100 cfu/ml and 102 cfu/ml respectively.For simulation samples before enrichment,the LDLs of MCO157APS,MACS and RTF-PCR were 105 cfu/ml,101 cfu/ml and 104 cfu/ml,while those for simulation samples after enrichment were 102 cfu/ml,100 cfu/ml and 100 cfu/ml respectively.Conclusions The detection limit of MACS in detecting Escherichia coli O157：H7 before and after enrichment culture is the lowest.The detection limit of RTF-PCR after enrichment culture also reaches the lowest.These two methods are suitable for fast diagnosis of food poisoning and food-borne diseases.MCO157APS can easily lead to determination miss.Organic combination of MACS and RTF-PCR can markedly improve detection rate and sensitivity.Moreover,strains can be easily obtained.