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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。
Objective To establish a specific mtLSU nested PCR assay for detection of Pneumocystis sp., And to study its detection effect and its sensitivity and specificity in the experimental model of Pneumocystis infection in order to provide a new clinical test for Pneumocystis colonization or infection method. Methods Based on the conserved sequences of Pneumocystis major mitochondrial mitochondria, nested PCR primers were designed and optimized to detect the neosporial PCR system of Pneumocystis sp. The immunosuppression-induced pneumocystis-infected rat model was used as the test object. The positive rate and coincidence rate of Pneumocystis sp. Were detected by mtLSU nested PCR and GMS staining. The positive samples were purified by DNA, and the molecular clones were constructed. The sensitivity of mtLSU nested PCR was detected by fold dilution method. The common pathogens of respiratory tract infection (Candida glabrata, Candida parapsilosis, Yeast, Candida albicans, Candida krusei, hemolytic streptococcus, Staphylococcus aureus, Mycoplasma) as a control to test the specificity of the method. Results Fourteen lungs of Pneumocystis aeruginosa were detected by GMS staining. The detection rate was 70.0% (14/20) in control group and 0 (0/20) in control group. The mtLSU nested PCR assay could detect the Pneumocystis sp. Gene in the experimental group, the positive rate was 100% (20/20), while the control group was negative. The positive rate of mtLSU nested PCR was significantly different from that of GMS staining (P <0.05). Sensitivity test results showed that the minimum amount of 366fg Pneumocystis gene was detected by mtLSU nested PCR. Specificity tests showed that the mtLSU nested PCR amplification results of the other 8 common respiratory pathogens were all negative. Conclusion The mtLSU nested PCR method for detecting Pneumocystis sp. Gene has been successfully established. The animal model test results show that this method is highly sensitive and specific and is expected to be used in the gene detection of Pneumocystis sp. In clinical specimens.