摘要目的 采用多重荧光PCR结合Allglo探针技术,建立一种快速、特异、灵敏的鉴定艰难梭菌相关毒素基因的方法,并对浙江杭州地区腹泻患者的产毒型艰难梭菌感染现状进行调查研究.方法 应用型研究.针对艰难梭菌毒素tcdA和tcdB基因设计引物和相应的Allglo探针,优化PCR扩增体系,并评价其特异性、灵敏度、重复性.收集2012年8至12月杭州市第一人民医院的门诊腹泻患者粪便标本共180份,对粪便中携带tcdA和tcdB基因的产毒型艰难梭菌进行鉴定,同时通过对tcdA和tcdB基因直接测序进一步验证荧光PCR检测结果的可靠性.结果 本试验建立的结合Allglo探针的多重荧光PCR方法可同时、准确、特异地鉴定艰难梭菌tcdA和tcdB基因,灵敏度可达10 CFU/ml.定量检测tcdA基因的循环阈值(Ct)值变异系数分别为2.30％、0.42％、0.81％,检测tcdB基因的Ct值变异系数分别为1.91％、1.02％、1.18％,均小于5％.在180份腹泻粪便样本中,检测出26份阳性样本,阳性率为14.4％,其中tcdA和tcdB毒素基因均阳性的样本为23份(23/26,88.5％),仅tcdA毒素基因阳性的样本为2份(2/26,7.7％);仅tcdB毒素基因阳性的样本为1份(1/26,3.8％).同时,采用直接测序方法对样本进行扩增检测和序列比对,与荧光定量PCR结果完全一致.结果显示浙江杭州地区腹泻患者感染的产毒型艰难梭菌以同时携带tcdA和tcdB毒素基因的菌株为主.结论 本研究建立的Allglo探针的多重荧光PCR方法可用于临床上快速、准确地检测产毒型艰难梭菌.

abstractsObjective To establish a rapid,specific and sensitive assay based on multiplex realtime PCR integrated with Allglo probes for detection and identification of Clostridium difficile related toxin genes,and investigate the situation of diarrhea patients infected by toxigenic C.difficile in Hangzhou.Methods The primer pairs and Allglo probes that were specific to tcdA,tcdB gene of C.difficile were designed,and then the PCR amplification condition was optimized,the specificity,low of limitation and reproducibility were evaluated.A total of 180 diarrheal stool specimens were collected from August to December 2012 from Hangzhou First People's Hospital.Finally,the multiplex real-time PCR results were compared with the results obtained by direct sequencing.Results The low of limitation was 10 CFU/ml.The coefficients of variation for tcdA gene were 2.30％,0.42％,0.81％ respectively,and the coefficients of variation for tcdB gene were 1.91％,1.02％,1.18％,respectively.All of them were less than 5％.There were 26 positive in 180 diarrhea stool specimens,the positive rate was 14.4％.Among the positive specimens,23 stool specimens were tcdA and tcdB positive (23/26,88.5％),and then only two stool specimens were tcdA positive to and tcdB negative (2/26,7.7％),only one stool specimen was tcdA negative and tcdB positive to was detected (1/26,3.8％).Then,all the specimens were detected by using a direct sequencing method,and the sequences were aligned with standard.The above results were in correspondence with the results of the real-time PCR.Conclusions The results indicated that it was the main characteristics for C.difficile strains with tcdA and tcdB toxin genes infecting diarrheal patients in Hangzhou,Zhejiang.The assay established here could be used in clinic for rapid,accurate detection of toxigenic C.difficile.