肺炎衣原体实时定量PCR检测方法的建立-东南大学学报医学版

肺炎衣原体实时定量PCR检测方法的建立

单位：广州军区广州总医院检验科,广东广州 510010

分类号：R725.6

出版年·卷·期（页码）：2012·31·第一期（43-46）

摘要：

目的:探讨实时定量PCR检测肺炎衣原体(chlamydia pneumonia, Cpn)的方法。方法:根据GenBank提供的肺炎衣原体基因序列,选取高特异性和保守型的区域进行引物设计,并建立实时定量PCR(real-time PCR, RT-PCR)的检测方法。同时用肺炎支原体、肺炎双球菌、流感病毒、副流感病毒以及腺病毒对Cpn引物的特异性进行分析。对呼吸道感染患者200个咽拭子样本和68个肺泡冲洗液样本进行检测,以荧光抗体检测法作对照,评价实时定量PCR检测Cpn的准确性。结果:Cpn PCR引物对肺炎支原体、肺炎双球菌、流感病毒、副流感病毒以及腺病毒均显示阴性,对Cpn显示为阳性,特异性良好;荧光实时定量PCR技术检测的准确性优于荧光抗体检测法。结论:荧光实时定量PCR技术检测Cpn体灵敏度高、周期短,可作为临床诊断Cpn的手段。

Objective: To investigate real-time PCR as a method to detect chlamydia pneumonia in clinical settings. Methods: Primers were designed according to conserved DNA region of chlamydia pneumonia from GenBank. Mycoplasma pneumonia, pneumococci, influenza B virus, para influenza virus and adenovirus were used to examine the specificity of primers. Two hundred and sixty-eight patients with respiratory tract infections were determined by real-time PCR and fluorescent antibody. Accuracy of measurement was compared between real-time PCR and fluorescent antibody. Results: Real-time PCR showed that primer specificity on Mycoplasma pneumonia, Pneumococci, Influenza virus, Para influenza virus and Adenovirus were negative but was positive on Chlamydia pneumonia. The primers were only specific for Chlamydia pneumonia. The diagnostic accuracy of real-time PCR was better than that of immunofluorescence. Conclusions: Real time quantitative PCR can be used as a clinical diagnostic assay with chlamydia pneumonia for high sensitivity and short period.

参考文献：

[1] KORPPI M,PALDANIUS M,HYVARINEN A,et al.Chlamydia pneumoniae and newly diagnosed asthma:a case-control study in 1 to 6-year-old children[J].Respirology,2004,9(2):255-259.
[2] 黄海辉,张婴元,汪复,等.亚洲地区肺炎支原体和肺炎衣原体在成人社区获得性肺炎中的流行病学研究[J].中国感染与化疗杂志,2008,8(2):89-93.
[3] SENN L,JATON K,FITTING J W,et al.Does Respiratory infection due to chlamydia pneumoniae Still Exist？[J].Clin Infect Dis,2011,53(8):847-848.
[4] 刘广超,吴移谋.肺炎嗜衣原体诊断方法的研究进展[J].微生物学免疫学进展,2006,34(4):71-75.
[5] 叶刚强.肺炎衣原体血清学检测及临床价值[J].国际医药卫生导报,2006,12(3):59.
[6] LI Y,WANG Y,NIE F,et al.A nested multiplex polymerase chain reaction assay for the differential identification of three zooanthroponotic chlamydial strains in porcine swab samples[J].J Vet Diagn Invest,2011,23(4):673-681.
[7] BRITTAIN L R,NORD S,OLOFSSON S,et al.Multiplex real-time PCR for detection of respiratory tract infections[J].J Clin Virol,2008,41(1):53-56.
[8] 方芳,窦常胜,浦春.外周血PCR和ELISA方法早期诊断婴幼儿Cpn感染[J].中华全科医学,2008,6(7):667-668.
[9] 郑广力,孙利炜,李丽红.荧光定量PCR在肺炎衣原体感染中的诊断评价[J].临床儿科杂志,2009,27(7):619-621.
[10] 宫道华.小儿感染病学[M].北京:人民卫生出版社,2003:3-5.
[11] APFALTER P,BAROUSCH W,NEHR M,et al.Comparison of a new quantitative ompA-based real-time PCR TaqMan assay for detection of chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays[J].J Clin Microbiol,2003,41(2):592-600.

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录