应用PCR检测产vero毒素大肠杆菌

Detection of Verotoxin producing Escherichia coli by the Polymerase Chain Reaction

根据产ｖｅｒｏ毒素大肠杆菌（ＶＴＥＣ）产生的ｖｅｒｏ毒素１（ＶＴ１）和ｖｅｒｏ毒素２（ＶＴ２）的基因序列，合成了２对寡核苷酸引物，建立了聚合酶链式反应（ＰＣＲ）扩增方法。共对４株产ｖｅｒｏ毒素大肠杆菌和其他７个属的４１株肠道致病菌中的ＶＴ基因进行了检测，结果仅从用传统组织培养方法确定为ＶＴ阳性的产ｖｅｒｏ毒素大肠杆菌和痢疾１型志贺氏菌提取的ＤＮＡ中观察到了ＰＣＲ扩增产物，而从其他肠道致病菌提取的模板ＤＮＡ中未扩增出任何ＤＮＡ片段。用这２对引物可以清楚地区分产ＶＴ１、ＶＴ２或ＶＴ１／ＶＴ２的大肠杆菌。ＰＣＲ扩增方法的检测敏感性为３２０ｐｇ全细菌ＤＮＡ，用ｖｔ１引物可以检测出低达３２ｐｇ的全细菌ＤＮＡ。

Two sets of synthetic oligonucleotide primers derived from sequences of the verotoxin 1 (VT1) and verotoxin 2 (VT2) genes were used in a polymerase chain reaction (PCR) amplification procedure to detect these genes in some enteric pathogens. A total of 4 verotoxin producing Escherichia coli strains and 41 other recognized pathogens were studied. PCR amplification products identifying the VT1 and VT2 gene sequences were observed only in DNA extracted from strains found to be VT positive by traditional tissue culture assays. The primers were able to distinguish clearly the VT1 and/or VT2 producing strains of Escherichia coli. Template DNA extracted from other enteric pathogens was found to be negative with the excepion of 1 strain of Shigella dysenteriae type 1 in which a good amplification with the VT1 primer was observed. The sensitivity of the PCR procedure for detection of both VT1 and VT2 genes was determined to be 320 pg of total cell DNA. Furthermore, the VT1 gene was easily detected when only 32 pg of DNA was used as a template in the PCR procedure.