| 高正琴,岳秉飞,贺争鸣.泰泽氏菌TaqManMGB探针荧光定量PCR方法的—立及应用[J].Chinese journal of Epidemiology,2012,33(2):226-228 |
| 泰泽氏菌TaqManMGB探针荧光定量PCR方法的—立及应用 |
| Development and application of TaqMan MGB probe fluorescence quantitative PCR method for rapid detection of Clostridium piliforme |
| Received:August 19, 2011 |
| DOI： |
| KeyWord: 泰泽氏菌 小沟结合物探针 荧光定量PCR |
| English Key Word: Clostridium piliforme Minor groove binder probe Fluorescence quantitative PCR |
| FundProject:中国食品药品检定研究院中青年研究发展基金(2010C5) |
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| Abstract: |
| 目的 建立泰泽氏菌的TaqMan小沟结合物(MGB)探针荧光定量PCR检测方法。方法 针对泰泽氏菌16SrRNA基因的保守区设计特异性引物和探针, 建立MGB探针荧光定量PCR方法, 并验证该方法的特异性、敏感性和稳定性。对2008—2011年采集的1156份临床样本进行检测, 同时进行普通PCR检测作为对照。结果 泰泽氏菌TaqManMGB探针荧光定量PCR方法具有高度特异性, 与肝螺杆菌、幽门螺杆菌、空肠弯曲菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌之间无交叉反应, 检测灵敏度达2.2copy/nl。标准曲线显示各浓度范围内具有良好线性关系, 相关系数为0.999, 斜率为-3.204, PCR效率为100%。对1156份临床样本进行检测, 荧光定量PCR检出101份泰泽氏菌阳性样本, 而普通PCR则仅检出44份。荧光定量PCR方法从临床样本中检出泰泽氏菌DNA仅需2h。结论 TaqManMGB探针荧光定量PCR方法具有特异、灵敏和稳定性, 适于泰泽氏菌的快速检测。 |
| English Abstract: |
| Objective To develop a TaqMan MGB probe-based, sensitive and specific fluorescence quantitative PCR assay method for rapid detection of Clostridium piliforme. Methods Primers and probes specific to 16S rRNA gene of Clostridium piliforme were designed. A TaqMan MGB probe-based, fluorescence quantitative PCR method was established. Specificity, sensitivity and stability of the method were assessed, followed by real-time quantitative PCR assay to detect Clostridium piliforme on 1156 clinical specimens during 2008-2011 and compared with conventional PCR assay. Results The specificity of TaqMan MGB probe-based fluorescence quantitative PCR was high and did not show cross-reactivity with Helicobacter hepaticus, Helicobacter pylori, Campylobacter jejuni, Pasteurella pneumotropica, Escherichia coli or Pseudomonas aeruginosa. The detection limit was 2.2 copies/jxl. The correlation coefficient and slope value of standard curve were 0.999 and -3.204, respectively and the efficiency of TaqMan MGB-based probe fluorescence quantitative PCR assay was 100%. When the TaqMan MGB-based probe fluorescence quantitative PCR assay was preformed to detect Clostridium piliforme on 1156 clinical specimens, a total of 101 specimens showed positive on Clostridium piliforme. However, only 44 specimens showed positive when conventional PCR was used. The real-time quantitative PCR for Clostridium piliforme could be completed within 2 hours. Conclusion The TaqMan MGB-based probe fluorescence quantitative PCR assay method was a reliable, specific, sensitive and useful tool for rapid detection of Clostridium piliforme. |
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