多重PCR法检测产志贺毒素性大肠杆菌的4个血清型

Determination of 4 serotypes of shiga toxin Escherichia coli by multiplex PCR assay

目的建立多重PCR法检测产志贺毒素性大肠杆菌(shiga toxin-producing Escherichia coli, STEC)主要血清型O26、O145、O45和O121的分析方法。方法根据GenBank登录序列,设计扩增O抗原翻转酶(wzx)基因的引物,建立PCR方法,并以STECO26、O145、O45和O121基因组为模板,检验多重PCR的灵敏度和特异性。使用建立的PCR,检测牛胴体表面拭子,阳性扩增条带送测序,以验证PCR扩增的可靠性。同时将阳性扩增样品,涂布显色平板,分离靶标血清型细菌。结果本研究成功建立STEC O26、O145、O45和O121的多重PCR方法, PCR循环参数中退火温度为60℃,扩增片段分别为249、353、890和587 bp。多重PCR直接检测O26、O145、O45和O121时,最低检测限介于10~3～10~4 CFU/mL,而增菌后再检测,最低检测限均为1CFU/m L。多重PCR用于其他血清型STEC,和非大肠杆菌扩增时,均未扩增出目的条带,只有O26、O145、O45和O121能够扩增出相应条带。当使用多重PCR直接检测胴体擦拭子时,阳性率为5.45%(3/55),主要为O26、O145血清型;增菌后检测阳性率为7.27%(4/55),主要为O26、O145和O121血清型。阳性PCR扩增样品,成功分离到O26两株、O145和O121各一株。分离菌株具有典型大肠杆菌的生化特性,携带STEC代表性毒力因子志贺毒素和紧密素,且具有多重耐药性。结论以STECO26、O145、O45和O121的wzx基因为检测靶标,成功建立多重PCR方法,灵敏度和特异性良好,与细菌分离联合使用,可减少工作量,精准分离目的病原菌。

Objective To establish a multiplex PCR method for determination of 4 predominant serotype O26,O145,O45 and O121 of shiga toxin-producing E.coli(STEC).Methods According to the GenBank accession sequence,the primers for amplifying the O antigen flipping enzyme(wzx) gene were designed,and the PCR method was established.The sensitivity and specificity of multiplex PCR were tested using the STEC O26,O145,O45 and O121 genomes as templates.Using the established PCR,the swabs on the surface of the bovine corpus callosum were detected,and the positive amplified bands were sent for sequencing to verify the reliability of the PCR amplification.At the same time,the sample was positively amplified,coated with a chromogenic plate,and the target serotype bacteria were isolated.Results This study successfully established STEC O26,O145,O45 and O121 multiplex PCR method and PCR cycle parameters in the annealing temperature was 60 ℃,fragments amplified 249,353,890 and 353 bp,respectively.When O26,O145,O45 and O121 were directly detected by multiple PCR,the minimum limit of detection was between 10~3 and 10~4 CFU/mL,while the minimum limit of detection was 1 CFU/mL after bacteria enrichment.When multiple PCR was applied to other serotypes of STEC and non-Escherichia coli,the target bands were not amplified,only O26,O145,O45 and O121 could amplify the corresponding bands.When the carcass swabs were directly detected by multiple PCR,the positive rate was 5.45%(3/55),mainly O26 and O145 serotypes.The positive rate after bacteria enrichment was 7.27%(4/55),mainly O26,O145 and O121 serotypes.Two strains of O26,one strain of O145 and one strain of O121 were isolated by positive PCR amplification.The isolated strain had the biochemical characteristics of typical Escherichia coli,carried STEC representative virulence factors shiga toxin and ghrelin,and had multi-drug resistance.Conclusion Using the wzx gene of STEC O26,O145,O45 and O121 as the detection target,the multiplex PCR method is successfully established,and the sensitivity and specificity are good.Combined with the separation of bacteria,the workload can be reduced and the pathogenic bacteria can be accurately separated.