肺孢子菌基因LAMP检测方法的建立

Development of the LAMP method for detecting the Pneumocystis

目的建立检测肺孢子菌基因的特异性环介导等温扩增(LAMP)方法,以期为临床检测肺孢子菌感染提供简便敏感的新方法。方法依据肺孢子菌核内核糖体小亚基16s rRNA保守序列设计特异性引物,建立检测肺孢子菌基因的LAMP和PCR反应体系。以实验感染肺孢子菌大鼠模型为检测对象,以倍比稀释的重组肺孢子菌基因组DNA质粒为模板,并与PCR方法对比,检验LAMP的敏感性;用呼吸道感染常见的7种病原体(光滑念珠菌、近平滑念珠菌、热带假丝酵母菌、白色念珠菌、溶血性链球菌、金黄色葡萄球菌MRSA株、支原体FH株)作对照,检测LAMP方法的特异性。结果本研究建立的LAMP法检测肺孢子菌基因的最低检出拷贝数为50 copies/ml,敏感性高于普通PCR最低检出拷贝数(104 copies/ml);检测肺孢子菌基因的特异性强,不与其他7种病原发生交叉反应。结论本研究成功地建立了特异性好,敏感性高的肺孢子菌基因LAMP检测方法。并显示检测程序简单,结果观察方便,且不需要特殊设备等优点。适用于临床肺孢子菌定植或感染的检测。

Objective To provide a new way for detecting the Pneumocystis infection. Methods The LAMP primers was designed basis on nuclear small subunit ribosomal conservative sequence;the LAMP system was established and optimized. Immunosuppression induced rat model of Pneumocystis infection was established. The genomic DNA was extracted and purified from the rat models infected with Pneumocystis; a recombinant plasmid was constructed. The sensitivity of LAMP was tested by multiple proportion dilute method and compared with the PCR methods. The specificity was examined by compared with Candida glabrata, Candida parapsilokis, Candida tropicalis,Candida krusei, Staphylococcus aureus, Mycoplasma pneumomiae and Streptococcus pneumoniae. Results The specific gene of Pneumocystis was detected by LAMP and shown no cross-reaction with the other strains. The lowest detection rate of LAMP detection of Pneumocystis gene was 50 copies / ml, and the lowest detection rate of PCR was 104 copies / ml. Conclusion LAMP method was developed successfully for detecting Pneumocystis with good specificity and sensitivity. The new method is now available for clinical detection of Pneumocystis.