沙眼衣原体荧光定量PCR检测方法的建立
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摘要
目的建立沙眼衣原体实时荧光定量PCR检测技术,以期用于沙眼衣原体的感染检测。方法针对沙眼衣原体ompA基因序列保守区域设计引物和TaqMan探针,构建标准质粒并制作标准曲线,建立沙眼衣原体实时荧光定量检测方法,通过对人型支原体等的检测评价方法的特异性。结果建立的沙眼衣原体荧光定量PCR体系能特异性检测沙眼衣原体,与人型支原体、解脲脲原体、淋球菌、大肠埃希菌EDL933、金黄色葡萄球菌无交叉反应;标准曲线在3.9×109～3.9×103 copies/μl之间线性关系良好(r2=0.99);方法的灵敏度高,检测最低拷贝数为3.9copies/μl;组内及组间变异系数均<5%。结论建立的检测沙眼衣原体实时定量PCR方法特异、灵敏,可用于沙眼衣原体感染的早期筛查和临床快速诊断。
Objective To create a real-time fluorescence quantitative PCR assay for detection of Chlamydia trachomatis. Methods Primers and TaqMan probes were designed in accordance with the conserved region of the ompA gene of C.trachomatis.The standard plasmid was constructed and a standard curve was constructed to create a TaqMan real-time fluorescent quantitative PCR assay to detect C.trachomatis.The specificity of the assay was evaluated by detecting pathogens such as Mycoplasma hominis. Results The assay specifically detected C.trachomatis and did not cross-react with Mycoplasma hominis,Ureaplasma urealyticum,Neisseria gonorrhoeae,E.coli EDL933,or Staphylococcus aureus.The standard curve exhibited good linearity(r2=0.99)between 3.9×109-3.9×103 copies/μl.The assay was highly sensitive,and its detection limit was 3.9copies/μl.Coefficients of variation within and between groups were all less than 5%. Conclusion The TaqMan real-time fluorescent quantitative PCR assay is a rapid,sensitive,and specific method for the quantitative detection of C.trachomatis and can be used in the early clinical diagnosis of C.trachomatis.
Objective To create a real-time fluorescence quantitative PCR assay for detection of Chlamydia trachomatis. Methods Primers and TaqMan probes were designed in accordance with the conserved region of the ompA gene of C.trachomatis.The standard plasmid was constructed and a standard curve was constructed to create a TaqMan real-time fluorescent quantitative PCR assay to detect C.trachomatis.The specificity of the assay was evaluated by detecting pathogens such as Mycoplasma hominis. Results The assay specifically detected C.trachomatis and did not cross-react with Mycoplasma hominis,Ureaplasma urealyticum,Neisseria gonorrhoeae,E.coli EDL933,or Staphylococcus aureus.The standard curve exhibited good linearity(r2=0.99)between 3.9×109-3.9×103 copies/μl.The assay was highly sensitive,and its detection limit was 3.9copies/μl.Coefficients of variation within and between groups were all less than 5%. Conclusion The TaqMan real-time fluorescent quantitative PCR assay is a rapid,sensitive,and specific method for the quantitative detection of C.trachomatis and can be used in the early clinical diagnosis of C.trachomatis.
引文
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[8] Van Der Pol B.Cobas?4800:a fully automated system for the detection of C.trachomatis and Neisseria gonorrhoeae[J].Expert Rev Mol Diagn,2014,13(2):131-40.
[9]郑铁洪,曾泰生,邱莉霞,等.深圳市福田区生殖道沙眼衣原体感染流行病学特征分析[J].公共卫生与预防医学,2016,27(4):50-3.
[10] Wei H,Zou S,Yang X,et al.Development of multiplex realtime quantitative PCR for simultaneous detection of C.trachomatis and Ureaplasma parvum[J].Clin Biochem,2012,45(9):663-7.
[11]刘丽君,凌继祖,赵福林.急性呼吸道感染患儿肺炎支原体和肺炎衣原体病原学特征分析[J].中国病原生物学杂志,2017,12(2):174-7,181.
[12] Pearce DM,Shenton DP,Holden J,et al.Evaluation of a novel electrochemical detection method for C.trachomatis:Application for point-of-care diagnostics[J].IEEE Trans Biomed Eng,2011,58(3):755-8.
[13]刘安元,李群,聂倩,等.沙眼衣原体pORF5质粒蛋白激活p38MAPK和NALP3信号通路调控THP-1细胞IL-1β分泌的研究[J].中国病原生物学杂志,2018,13(2):146-51.
[14] Hoque SM,Hossain MA,Paul SK,et al.Detection of C.trachomatis by immunological and genetic methods in female sex workers and the local female population of reproductive age in Mymensingh,Bangladesh[J].Jpn J Infect Dis,2013,66(3):256-9.
[15] van Dommelen L,Wolffs PF,van Tiel FH,et al.Influence of temperature,medium,and storage duration on C.trachomatis DNA detection by PCR:Table 1[J].Clin Microbiol,2013,51(3):990-2.
[2] Moncada J,Shayevich C,Philip SS,et al.Detection of C.trachomatis and Neisseria gonorrhoeae in rectal and oropharyngeal swabs and urine specimens from men who have sex with men with Abbott?s M2000realtime[J].Sexually Transm Dis,2015,42(11):650-1.
[3] Doseeva V,Forbes T,Wolff J,et al.Multiplex isothermal helicase-dependent amplification assay for detection of C.trachomatis and Neisseria gonorrhoeae[J].Diagn Microbiol Infect Dis,2011,71(4):354-5.
[4] Sood S,Satpathy G,Kapil A,et al.The role of a commercial enzyme immuno assay antigen detection system for diagnosis of C.trachomatis in genital swab samples[J].Indian Med Microbiol,2011,29(4):411-3.
[5] Lehmusvuori A,Juntunen E,Tapio A,et al.Rapid homogeneous PCR assay for the detection of C.trachomatis in urine samples[J].J Microbiol Methods,2010,83(3):302-6.
[6] Kr?lov K,Frolova J,Tudoran O,et al.Sensitive and Rapid Detection of C.trachomatis by recombinase polymerase amplification directly from urine samples[J].Mol Diagn,2014,16(1):127-35.
[7]Miller A,Bromhead C,Jones M,et al.Mucus digestion improves the detection of C.trachomatis and Neisseria gonorrhoeae on the cobas 4800[J].Sex Transm Dis,2012,39(9):733-4.
[8] Van Der Pol B.Cobas?4800:a fully automated system for the detection of C.trachomatis and Neisseria gonorrhoeae[J].Expert Rev Mol Diagn,2014,13(2):131-40.
[9]郑铁洪,曾泰生,邱莉霞,等.深圳市福田区生殖道沙眼衣原体感染流行病学特征分析[J].公共卫生与预防医学,2016,27(4):50-3.
[10] Wei H,Zou S,Yang X,et al.Development of multiplex realtime quantitative PCR for simultaneous detection of C.trachomatis and Ureaplasma parvum[J].Clin Biochem,2012,45(9):663-7.
[11]刘丽君,凌继祖,赵福林.急性呼吸道感染患儿肺炎支原体和肺炎衣原体病原学特征分析[J].中国病原生物学杂志,2017,12(2):174-7,181.
[12] Pearce DM,Shenton DP,Holden J,et al.Evaluation of a novel electrochemical detection method for C.trachomatis:Application for point-of-care diagnostics[J].IEEE Trans Biomed Eng,2011,58(3):755-8.
[13]刘安元,李群,聂倩,等.沙眼衣原体pORF5质粒蛋白激活p38MAPK和NALP3信号通路调控THP-1细胞IL-1β分泌的研究[J].中国病原生物学杂志,2018,13(2):146-51.
[14] Hoque SM,Hossain MA,Paul SK,et al.Detection of C.trachomatis by immunological and genetic methods in female sex workers and the local female population of reproductive age in Mymensingh,Bangladesh[J].Jpn J Infect Dis,2013,66(3):256-9.
[15] van Dommelen L,Wolffs PF,van Tiel FH,et al.Influence of temperature,medium,and storage duration on C.trachomatis DNA detection by PCR:Table 1[J].Clin Microbiol,2013,51(3):990-2.