液相芯片鉴别检测7种出血性大肠埃希菌血清型

Identification and Serotyping of Seven Shiga Toxin-producing Escherichia coli(STEC)Serogroups with Luminex High Throughput Assays

为了建立一种同时检测O157、O26、O45、O103、O111、O121和O145等7种血清型的出血性大肠埃希菌的高通量检测方法,通过筛选比对大肠埃希菌O157血清型的fbE基因,O26、O103和O111血清型的WZX基因,O45、O121和O145血清型的WZY基因,设计了针对各个血清型的特异性引物和探针。通过在所有的特异性引物5′端加入超级引物的策略,达到了通过一次PCR反应,同时多重扩增7个血清型的7个目标片段的效果。将加尾多重扩增与液相芯片高通量检测相结合,建立了同时检测7种血清型的出血性大肠埃希菌的液相芯片检测方法,并对方法检测体系及反应条件进行了优化。所建立的7种出血性大肠埃希菌液相芯片检测方法灵敏,其灵敏度与荧光PCR的灵敏度相差100倍。所建立的方法特异,单个血清型的探针与相应的PCR扩增的阳性产物之间均有特异性的杂交,LQRR值介于17~63之间。7种血清型的混合探针与各个血清型菌株的PCR与扩增的阳性产物之间均有特异性的杂交,LQRR值介于11~23之间,与其他血清型出血性大肠埃希菌和非出血性大肠埃希菌均无交叉反应。试验结果表明,所建立的液相芯片检测大肠埃希菌O157、O26、O45、O103、O111、O121和O145等7个血清型的检测方法具有快速、特异性强、灵敏度高等特点,可用于主要出血性大肠埃希菌的快速鉴别检测。

This study developed a multiplex PCR integrated with luminex technology to rapidly sreotype simultaneously seven Shiga toxin-producing Escherichia coli （STEC） serogroups including O157,026, O45,O103,O111,O121 and O145. Primers and probes were designed to amplify rfbE gene of O157,WZX genes of O26,O103 and Ol11, WZY genes of O45, O121 and O145. Universal super primers were intro- duced to establish a multiplex PCR to amplify all seven targets together,and then the multiplex PCR prod- ucts were further tested by Luminex assay with probes, combined to give a STEC rapid high throughput serotyping test. The multiplex PCR tailed with universal super primers could amplify all seven specific tar- gets at one time. Mono probe with one STEC serogroup could hybrid specifically with its PCR products, and Luminex qualitative ratio results （LQRR） were among 17-63. Probes of seven sebogroups were mixed together and tested with Luminex assay and the LQRR were among 11-23. The rapid high throughput sero- typing test presents the following features： high throughput, able to determine the serogroups of 7 target genes in O157,O26,O45,O103,O111,O121 and O145 of the STEC at one time;rapid,completing the sero- typing within 6 hours; high specificity, ensured the specificity of the different serogroups by using two primers and one probe,no cross reaction occurring between the serogroups, no non-specific reactions with other STEC serogroups and bacteria,and high sensitivity. The rapid high throughput serotyping test can satisfy the needs of STEC rapid testing.