聚合酶链反应检测出血性大肠埃希菌

为建立一种针对出血性大肠埃希菌的特异性聚合酶链反应（ＰＣＲ）诊断方法，根据Ｏ１５７：Ｈ７血清型大肠埃希菌ｈｌｙＡＢ基因的ＤＮＡ序列，设计８个引物，使用３２株Ｏ１５７：Ｈ７血清型。结果表明，引物ＲＨ２８－ＲＨ３０和ＲＨ２６－ＲＨ３１敏感性和特异性好，达１００％。ＲＨ２６－ＲＨ３１产物的分子量较大，为２ｋｂ。ＲＨ２８－ＲＨ３０的产物为３３８ｂｐ，比较适合检验使用。研究还发现，使用煮沸法制备ＰＣＲ模板，方法简便、快速、实用，且对ＰＣＲ反应的特异性和敏感性无影响。ｈｌｙＡＢ基因片段作为出血性大肠埃希菌ＤＮＡ探针的特异性和敏感性是明显的，且实用性好。根据出血性大肠埃希菌独特的ｈｌｙＡＢ基因序列设计的ＰＣＲ引物，在理论和实际应用方面均有其优越性。

Development of a specific PCR method for detection of entero-hemorrkagic Escherichia coli

ntero-hemorrhagic Escherichia coli is a diarrhea-inducing pathogen discovered recently. Based onthe hlyAB gene sequence of E. coli O1 57:H7,a PCR method for detection of entero-hemorrhagic E.coli was developed.With eight primers designed and 3 2 strain of E. coli O 1 5 7:H 7,the best resultswere obtained with primer pair of RH 28-RH 30 and RH26-RH 3 1,with sensitivity and specificity of100%. Since the PCR product for RH26-RH31 and RH28-RH30 were 2kb and 338bp respectively,RH28-RH30 is most suitable for detection and diagnosis purpose. We also observed that the boilingmethod for preparing template is simple,practical and rapid,without affecting the specificity and sensi-tivity. Since EHEC specific probe pCVD419 tested hlyAB genes has been proved to be very specific andsensitive by many laboratories in the world,the PCR method developed by us has advantages on detec-tion of EHEC strains in comparison with other method based on SLTs gene sequence.