川西北牦牛肉中产志贺毒素大肠杆菌的分离鉴定及stx2基因的序列分析

Isolation and Identification of Shiga Toxin-producing Escherichia coli from Yak(Bos grunniens)in the Northwestern Sichuan,and Sequence Analysis of stx2Gene

为了解中国川西北牦牛肉中产志贺毒素大肠杆菌(STEC)的携带情况及stx2的亚型和特征,试验将采集的204份川西北牦牛肉样品(各25g)增菌培养后,每份挑取5个可疑菌落,采用stx1、stx2双重PCR方法检测STEC,对分离株中的stx2分型并克隆测定stx2编码区序列。结果显示,在204份样品中分离出8株STEC,平均分离率为3.9%(8/204);存在4个不同的O血清型,分别为O38(4)、O50(1)、O74(2)、O150(1);在6株含有stx2的菌株中,其中有2株为stx2a型、4株为stx2c型。结果表明牦牛源分离株氨基酸序列与人源和牛源菌株同源性较高;由stx2A、B亚基的氨基酸序列系统进化树可知,牦牛源分离株与人源、牛源菌株聚为一支,表明它们之间遗传距离相对较近,牦牛源stx2各自分布在自己的小分支中,表明牦牛源STEC stx2与人源和牛源stx2相比,尽管亲缘关系较近,但仍存在一定程度的差异。

In order to study the prevalence of yak meat Shiga toxin-producing Escherichia coli （STEC） in Northwestern Sichuan and its stx2 subtype,five suspected colonies were picked from each enrichment culture of 204 collected yak meat samples （25 g）. Double PCR assay was used to detect stxl and stx2,stx2 was subtyped and its CDS region was sequenced. 8 STEC strains were isolated from 204 samples,in which the isolating rate of STEC was 3.9% （8/204）. 4 different serotypes O detected were O38（4） ,O50（1） ,O74（2） and O150（1） ,respectively. 6 strains were found carry stx2,and 2 strains were stx2a and 4 strains were stx2c. The results revealed that yak-origi- nal isolated strains had a high degree of amino acid homology with human-original and cattle-original strains; Amino acid phylogenetic trees of stx2 A and B subunits showed that yak-original, hu man-original and cattle-original strains gathered into the same clade,indicating that their genetic distance was relatively near. Yak-original stx2 were distributed in their own little branch, compared with the human-original and cattle-original strains,although yak-original STEC stx2 genetic relations were closer with them,but still had some differences.