大肠杆菌O120 O-抗原基因簇的破译及特异分子标识的鉴定

Sequence of Escherichia coli O120 O-antigen gene cluster and identification of molecular markers specific to O120

目的完成产志贺毒素大肠杆菌O120 O-抗原基因簇的破译,筛选和鉴定检测用特异分子标识。方法利用鸟枪法进行O-抗原基因簇序列的测定,进行生物信息学方法分析发现基因并预测基因功能,利用PCR方法筛选针对大肠杆菌O120特异基因和特异引物,利用基因芯片方法筛选特异探针。结果O-抗原基因簇序列全长12 485 bp,共含有10个基因:dTDP-鼠李糖合成途径基因(rmlB、rmlD、rmlA和rmlC),O-抗原转运酶基因(wzx),O-抗原聚合酶基因(wzy),3个糖基转移酶基因和1个功能未确定的基因。另外筛选和鉴定了2个特异基因、4对特异引物和6条特异探针,在模拟样品的鉴定中得到验证。结论多种特异分子标识可从样品中特异地检测出大肠杆菌O120,具有快速、灵敏和准确进行分子分型的优点。

Objective The O-antigen gene cluster of the E. ooli O120 representative strain was sequenced, 10 open reading frames （ORF） were assigned for functions on the basis of homology. Specific genes, primers and probes for E. coli O120 were identified. Methods The O-antigen gene cluster of E. coli O120 was amplified by long-range PCR and sequenced. By polymerase chain reaction and DNA microarray assay against representative stains for the all 166 E. coli and 43 Shigella O serotypes, 2 genes, 4 primer pairs and 6 oligonucleotide specific probes to E. coli O120 were identified. Results A sequence of 12 485 bases from galF to gnd was obtained, which contained 10 ORF, including genes coding for biosynthesis of dTDP-rhamnose （ rmlB, rmlD, rmlA, and rmlC）, glycosyl transferase genes, O-unit flippase gene （urzx） and O-antigen polymerase gene（ urzy）. The detection sensitivity of the specific PCR assay was 10 pg genomie DNA, 10 CFU/25 g in mock beef sample, or 103 CFU/g in mock stool specimens. Conclusion PCR and the DNA microarray assays established in this study can be used to identify E. coli O120 strains and may also be used to detect E. coli O120 strains in food, stool and other environmental samples.