肺炎衣原体PCR、套式PCR评价与临床应用

Comparison of PCP and Nested PCR Detected of Chlamydia Pneumoniae and Application

目的评价PCR和套式PCR检测肺炎衣原体(Chlamydia pneumoniae,Cpn)的特异性和灵敏度。方法按Campbell等建立PCR反应体系,按Tong等与秦玲等建立套式PCR反应体系,本文并以Cpn 16SrRNA基因(16SrDNA)为靶基因自行建立套式PCR反应体系,对Cpn、沙眼衣原体、鹦鹉热衣原体、大肠埃希稀菌、肺炎克雷伯菌、流感嗜血杆菌、粘质沙雷菌、洛菲不动杆菌、鲍曼不动杆菌、嗜肺军团杆菌、巨细胞病毒、单纯疱疹病毒进行检测以评价各方法的特殊性异性;以纯化的Cpn-DNA和1例阳性临床标本作梯度稀释后分别参与各反应体系检测以评价各方法的灵敏度。结果1、PCR和套式PCR均只能检出Cpn TW-183株和CWL-29株,而其它衣原体、支原体、细菌、病毒均不能检出;2、套式PCR检测Cpn较PCR敏感100～1000倍;3、经以16SrRNA基因为靶基因套式PCR检测新生儿肺炎(非吸入性肺炎)和儿童肺炎者咽拭子标本Cpn阳性率分别为16.2％和22.0％,肺结核者痰标本为18.9％,男性STD者尿道拭子标本为0％。结论 PCR和套式PCR检测Cpn均有较高的特异性,其中套式PCR比PCR敏感100～1000倍,在肺部炎症者咽拭子或痰标本中Cpn有较高的检出率。

Objective A specific and sensitive test for PCR and nested PCR (nPCR) detection of C. pneumoniae. Methods PCR are based on Campbell's reaction systems, nPCR are based on Tong' and Qin' reaction systems. This paper nPCR reaction ware performed using a set of nested primers defined from the 16S rRNA gene. C.pneumoniae, C.trachomatis, C.psittaci, E.coli, K.pneumoniae, H.infiuenzae, S. marcescens, A.lwoffi, A.baumannii, L.pneumoniae, CMV and HSV were detected. Results 1、The PCR and nPCR reacted with C.pneumoniae (TW-183 strain and CWL-29 strain) and gave no amplification prod- uct with other microorganisms; 2,The detection limit of nPCR was determind to be 5fg c.pneumoniae DNA. The sensitivity of the nPCR was about 100-1000 times higher than that of PCR; 3、Positive rates of nPCR of 16SrRNA gene were 16.2% in neonatal pneumonia, 22.0% in children pneumonia, 18.9% in TB patients and 0% in STD patients. Conclusion Specificity of PCR and nPCR was high, The sensitivity of the nPCR was higher than PCR