检测肺孢子菌巢式PCR方法的建立及其敏感性和特异性研究

Establishment of a nested PCR technique to detect Pneumocystis and study of its sensitivity and specificity

目的建立检测肺孢子菌基因的特异性mtLSU巢式PCR方法,研究其在实验动物肺孢子菌感染模型的检测效果及其敏感性和特异性,以期为临床提供检测肺孢子菌定植或感染的新方法。方法依据肺孢子菌线粒体大亚基保守序列设计巢式PCR内、外引物,建立并优化检测肺孢子菌基因的巢式PCR体系。以免疫抑制诱导肺孢子菌感染大鼠模型为检测对象,比较mtLSU巢式PCR与六亚甲基四胺银(GMS)染色法检测肺孢子菌的阳性率及符合率。对阳性标本进行DNA纯化,构建分子克隆,采用倍比稀释法检测mtLSU巢式PCR的敏感性;以呼吸道感染常见的8种病原体(光滑假丝酵母菌、近平滑假丝酵母菌、热带假丝酵母菌、白色念珠菌、克柔假丝酵母菌、溶血性链球菌、金黄色葡萄球菌、支原体)为对照,检测方法的特异性。结果肺印片GMS染色镜检实验组大鼠,14只查到肺孢子菌包囊,检出率为70.0%(14/20),对照组检出率为0(0/20)。用mtLSU巢式PCR均能检测实验组大鼠肺孢子菌基因,阳性率为100%(20/20),对照组均为阴性。mtLSU巢式PCR法阳性率与GMS染色法比较差异有统计学意义(P<0.05)。敏感性检验结果表明,mtLSU巢式PCR检测最小量为366fg的肺孢子菌基因。特异性检测显示,其他8种呼吸道常见病原体的mtLSU巢式PCR的扩增结果均为阴性。结论成功建立了检测肺孢子菌基因的mtLSU巢式PCR方法,动物模型检测结果表明该方法的敏感性高、特异性强,有望应用于临床患者标本中肺孢子菌的基因检测。

Objectives mtLSU（mitochondrial large-subunit）nested PCR was used to detect Pneumocystis genes in order to provide a new way to detect Pneumocystisinfection,and its sensitivity and specificity were tested in laboratory animals. Methods Nested PCR primers were designed in accordance with the conserved sequence of the Pneumocystis mitochondrial large subunit（mtLSU）gene.A nested PCR technique was established and optimized.The rate of detection and concordance of mtLSU nested PCR were tested in a rat model of Pneumocystis infection and these rates were compared to those of Gomori Methenamine Silver staining.Negative samples were subjected to DNA purification,a molecular clone was created,and the sensitivity of mtLSU nested PCR was tested using serial dilution.Specificity was examined by comparing Pneumocystis to Candida glabrata,Candida parapsilosis,Candida tropicalis,Candida krusei,Candida albicans,Staphylococcus aureus,Mycoplasma pneumoniae,and Streptococcus pneumoniae. Results Pneumocystis cysts was detected by GMS at a rate of 70.0%（14/20）and by mtLSU PCR at a rate of 100%（20/20）.The control group tested negative according to both GMS and PCR.Differences between mtLSU nested PCR and GMS staining in terms of the rate of detection were statistically significant（P〈0.05）.The concordance rate was 100% with GMS staining.Results of the susceptibility test indicated that the detection limit for mtLSU nested PCR was 366fg.mtLSU nested PCR was specific and yielded negative results for all of the 8other common respiratory pathogens. Conclusion An mtLSU nested PCR technique to detect Pneumocystis genes was successfully established.Testing in an animal model revealed that nested PCR had a higher sensitivity and specificity than GMS,and nested PCR might be useful when testing clinical specimens for Pneumocystis genes.