| 邵冬华,季娜,梁国威,刘静.TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立[J].中华流行病学杂志,2014,35(5):576-580 |
| TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立 |
| Using the real-time PCR assay to establish TaqMan-MGB probe for rapid identification of Clostridium difficile and its toxin |
| 收稿日期:2013-11-18 出版日期:2014-09-17 |
| DOI：10.3760/cma.j.issn.0254-6450.2014.05.024 |
| 中文关键词: 难辨梭菌 TaqMan-MGB探针 实时荧光定量PCR 菌属基因 毒素基因 |
| 英文关键词: Clostridium difficile TaqMan-MGB probe Real-time PCR Bacteria genus gene Toxin gene |
| 基金项目: |
|
| 摘要点击次数: 2922 |
| 全文下载次数: 1890 |
| 中文摘要: |
| 目的 建立一种简便、快速难辨梭菌菌属鉴定及其毒素基因筛查的荧光定量PCR检测方法。方法 采用TaqMan-MGB探针，通过real-time PCR分析系统，同时检测难辨梭菌菌属基因磷酸丙糖异构酶（Tpi）、A毒素（TcdA）、B毒素（TcdB）及缺失部分基因的A毒素（TcdAT），从特异度、灵敏度及其抗干扰性等方面评价该方法，并联合全自动酶联荧光免疫系统（VIDAS）检测50 例临床不明原因腹泻病例粪便标本探讨其应用价值。结果 难辨梭菌非产毒株 Tpi 基因（tpi）的检测下限是6×10-2 CFU/μl，产毒株 tpi、tcdA、tcdB、tcdAT 的检测下限为 6×10-1 CFU/μl；难辨梭菌非产毒株tpi检测下限批内、批间变异率分别为2.1%和2.3%；产毒株tpi、tcdA、tcdB、tcdAT的检测下限批内、批间变异率依次为3.0%和3.4%、2.9%和3.2%、5.3%和5.7%、2.7%和2.8%。临床常见分离菌株及梭菌属其他细菌对检测无干扰。50 例不明原因腹泻病例粪便标本中，采用TaqMan-MGB 探针实时荧光 PCR 与 VIDAS 酶标免疫检测 39 例阴性标本其符合率为 100%；6 例两方法检测均为阳性；3例VIDAS酶标免疫检测为可疑及2例为阴性，经TaqMan-MGB探针实时荧光PCR检测为A-/B＋菌株。结论 建立的TaqMan-MGB探针实时荧光定量PCR具有高通量、高灵敏度和重复性好的特性，且可筛查携带缺失部分基因的TcdA难辨梭菌菌株。 |
| 英文摘要: |
| Objective To develop a real-time PCR assay for the rapid identification of Clostridium（C.） difficile and its toxin. Methods TaqMan real-time PCR was developed for the rapid identification of species specific gene（tpi）of C. difficile strains and the toxins A（TcdA），B（TcdB） and truncated toxin A（TcdAT）. Sensitivity，specificity and anti-interference ability of these methods were estimated，as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay. Results The detection limits of tpi were 6×10-2 CFU/μland 6 × 10-1CFU/μ l in the non-oxin producing and toxin producing strains，respectively. The coefficients of variability（CV）of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3% . The CVs of intra-assay and inter-assay for the detection limit of tpi，tcdA，tcdB and tcdAT in the toxin producing strain were 3.0% and 3.4%，2.9% and 3.2%，5.3% and 5.7%，2.7% and 2.8%，respectively. No interferance was detected from other genus or species in clostridium. From 50 clinical samples，thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique，in which 3 were dubious and 2 were negative under VIDAS. Conclusion The newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis，clinically. |
|
查看全文
Html全文
查看/发表评论 下载PDF阅读器 |
|
| 关闭 |
