中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2014年
1期
32-35
,共4页
金大智%罗芸%罗丽%张政%叶菊莲%张严峻%吴方%方小飞%李辉
金大智%囉蕓%囉麗%張政%葉菊蓮%張嚴峻%吳方%方小飛%李輝
금대지%라예%라려%장정%협국련%장엄준%오방%방소비%리휘
梭菌,难辨%细菌毒素类%肠毒素类%细胞毒素类%荧光%聚合酶链反应
梭菌,難辨%細菌毒素類%腸毒素類%細胞毒素類%熒光%聚閤酶鏈反應
사균,난변%세균독소류%장독소류%세포독소류%형광%취합매련반응
Clostridium difficile%Bacterial toxins%Enterotoxins%Cytotoxins%Fluorescence%Polymerase chain reaction
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.
目的 採用多重熒光PCR結閤Allglo探針技術,建立一種快速、特異、靈敏的鑒定艱難梭菌相關毒素基因的方法,併對浙江杭州地區腹瀉患者的產毒型艱難梭菌感染現狀進行調查研究.方法 應用型研究.針對艱難梭菌毒素tcdA和tcdB基因設計引物和相應的Allglo探針,優化PCR擴增體繫,併評價其特異性、靈敏度、重複性.收集2012年8至12月杭州市第一人民醫院的門診腹瀉患者糞便標本共180份,對糞便中攜帶tcdA和tcdB基因的產毒型艱難梭菌進行鑒定,同時通過對tcdA和tcdB基因直接測序進一步驗證熒光PCR檢測結果的可靠性.結果 本試驗建立的結閤Allglo探針的多重熒光PCR方法可同時、準確、特異地鑒定艱難梭菌tcdA和tcdB基因,靈敏度可達10 CFU/ml.定量檢測tcdA基因的循環閾值(Ct)值變異繫數分彆為2.30％、0.42％、0.81％,檢測tcdB基因的Ct值變異繫數分彆為1.91％、1.02％、1.18％,均小于5％.在180份腹瀉糞便樣本中,檢測齣26份暘性樣本,暘性率為14.4％,其中tcdA和tcdB毒素基因均暘性的樣本為23份(23/26,88.5％),僅tcdA毒素基因暘性的樣本為2份(2/26,7.7％);僅tcdB毒素基因暘性的樣本為1份(1/26,3.8％).同時,採用直接測序方法對樣本進行擴增檢測和序列比對,與熒光定量PCR結果完全一緻.結果顯示浙江杭州地區腹瀉患者感染的產毒型艱難梭菌以同時攜帶tcdA和tcdB毒素基因的菌株為主.結論 本研究建立的Allglo探針的多重熒光PCR方法可用于臨床上快速、準確地檢測產毒型艱難梭菌.
목적 채용다중형광PCR결합Allglo탐침기술,건립일충쾌속、특이、령민적감정간난사균상관독소기인적방법,병대절강항주지구복사환자적산독형간난사균감염현상진행조사연구.방법 응용형연구.침대간난사균독소tcdA화tcdB기인설계인물화상응적Allglo탐침,우화PCR확증체계,병평개기특이성、령민도、중복성.수집2012년8지12월항주시제일인민의원적문진복사환자분편표본공180빈,대분편중휴대tcdA화tcdB기인적산독형간난사균진행감정,동시통과대tcdA화tcdB기인직접측서진일보험증형광PCR검측결과적가고성.결과 본시험건립적결합Allglo탐침적다중형광PCR방법가동시、준학、특이지감정간난사균tcdA화tcdB기인,령민도가체10 CFU/ml.정량검측tcdA기인적순배역치(Ct)치변이계수분별위2.30％、0.42％、0.81％,검측tcdB기인적Ct치변이계수분별위1.91％、1.02％、1.18％,균소우5％.재180빈복사분편양본중,검측출26빈양성양본,양성솔위14.4％,기중tcdA화tcdB독소기인균양성적양본위23빈(23/26,88.5％),부tcdA독소기인양성적양본위2빈(2/26,7.7％);부tcdB독소기인양성적양본위1빈(1/26,3.8％).동시,채용직접측서방법대양본진행확증검측화서렬비대,여형광정량PCR결과완전일치.결과현시절강항주지구복사환자감염적산독형간난사균이동시휴대tcdA화tcdB독소기인적균주위주.결론 본연구건립적Allglo탐침적다중형광PCR방법가용우림상상쾌속、준학지검측산독형간난사균.
Objective 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.
