荧光定量PCR和环介导等温扩增方法检测隐球菌CAP10基因的比较研究

Establishment of real-time PCR and loop-mediated isothermal amplification for detecting Cryptococcus neoformans CAP10 gene

目的建立荧光定量PCR和环介导等温扩增技术(LAMP)检测隐球菌荚膜相关蛋白基因(CAP10)的方法,并比较和评价两种方法的优缺点。方法针对新型隐球菌CAP10基因序列,使用在线软件设计引物,并构建质粒标准品,优化反应条件后用荧光定量PCR和LAMP两种方法检测定量的质粒,分析其敏感性和特异性,并检测临床标本。结果荧光定量PCR检测CAP10质粒的敏感性为6.8×101拷贝,LAMP方法的敏感性为6.8×103拷贝,PCR检测阳性率比LAMP方法高。两种方法特异性好,对脑膜炎奈瑟菌、白色念珠菌、热带念珠菌、黄曲霉、黑曲霉和大肠埃希菌的检测结果均为阴性。结论成功建立了检测CAP10基因的荧光定量PCR方法敏感性和特异性高,但是需要特殊仪器;LAMP方法敏感性较低,但其操作简单,无需特殊仪器和设备,加入核酸染料即能观察结果。两者均适用于新型隐球菌感染的检测。

Objective To establish real-time PCR and loop-mediated isothermal amplification （LAMP） systems for detecting Cryptococcus neoformans CAP10 gene. Methods Specific primers were designed targeting CAP10 gene of Cryptococcus neoformans, and the plasmid was constructed. After optimization of the reaction condition, the plasmid was quantitatively detected using real-time PCR and LAMP, and the detection sensitivity and specificity were evaluated. Clinical samples were also detected using the two methods. Results The detection sensitivity of real-time PCR and LAMP was 6.8×101 and 6.8×103 copies, respectively. Real-time PCR yielded a higher positivity rate than LAME Both of the two methods showed a high detection specificity and produced negative results in the detection of Neisseria meningitidis, Candida albicans, Candida tropicalis, Aspergillus Jlavus, Aspergillus niger and Escherichia coli. Conclusion Real-time PCR is highly sensitive and specific for detecting Cryptococcus neoformans CAP10 gene but requires sophisticated equipment. LAMP, though with a relatively lower sensitivity, is simple to operate without the need of special equipment, and the result can be conveniently observed. Both of the two methods are suitable for detecting Cryptococcus neoformans and evaluating the treatment outcomes.