艰难梭菌快速分子诊断方法的建立

Devising and clinical use of methods for rapid molecular detection of Clostridium difficile

目的建立用于快速检测艰难梭菌感染的聚合酶链式反应(PCR)和环介导等温扩增(LAMP)方法。方法收集有艰难杆菌感染症状患者的粪便样品,经CCFA选择性培养基筛选和VITEK MS飞行时间质谱仪分析获得艰难梭菌菌株;利用BLAST分析得到艰难梭菌特异性基因,以此为模板建立、优化并验证艰难梭菌PCR检测和LAMP检测方法。结果筛选得到3株产毒艰难梭菌,经数据分析获得艰难梭菌的两个特异性基因FliM和假定蛋白基因(hypothetical protein)。分别建立Flim基因的PCR检测方法和检测假定蛋白基因的LAMP方法,检测下限均达到了100个质粒/μl,且无非特异性扩增。结论 PCR法和LAMP法成本低,快速灵敏、特异性好,可用于艰难梭菌感染的快速检测。

Objective To use a polymerase chain reaction(PCR) and loop-mediated isothermal amplification(LAMP) to create two rapid methods for clinical detection of Clostridium difficile. Method Fecal samples were collected from patients with symptoms of a C. difficile infection. C. difficile strains were determined using CCFA selective medium screening and VITEK MS time-of-flight mass spectrometry analysis. C. difficile-specific genes were identified using BLAST analysis and then used as templates to create, optimize, and validate PCR and LAMP techniques for detection of C. difficile. The genomic DNA of several standard strains and the genome of C. difficile were used as templates to evaluate the specificity of PCR and LAMP. A recombinant plasmid containing specific genes of C. difficile was constructed, and the recombinant plasmid was serially diluted 10-fold to yield 7 concentrations. Two μl of the plasmid was used as a template to evaluate the sensitivity of PCR and LAMP. Results Three strains of toxin-producing C. difficile were screened. An analysis of the data revealed two specific FliM genes and a gene encoding hypothetical proteins of C. difficile. A PCR technique was devised to detect the Flim gene, and a LAMP technique was devised to detect the hypothetical protein gene. The detection limit was 100 plasmids/μl, and there were no nonspecific amplification bands. The above results indicated that the techniques devised in this study were specific and sensitive. Conclusion The use of PCR and LAMP to detect C. difficile is an inexpensive and quick approach with a high level of sensitivity and specificity. This technique can be used to rapidly detect C. difficile in clinical settings.