基因芯片技术检测性传播泌尿生殖道感染常见病原体的研究
摘要
近年来，性传播泌尿生殖道感染(包括淋病和非淋菌性尿道炎)的发病率在我国逐年增加，尤其是非淋菌性尿道炎(nongonococcal urethritis，NGU)，由于致病病原体种类多，具有潜伏感染和无症状排菌等生物学特性，给人类公共健康带来严重影响。
研究已证实淋病双球菌、解脲脲原体、沙眼衣原体、人型支原体、生殖支原体、单纯疱疹病毒Ⅰ(HSV-1)、单纯疱疹病毒Ⅱ(HSV-2)、白念珠菌及阴道毛滴虫为引起性传播泌尿生殖道感染(Sexually Transmitted Urogenital Infections，STUI)的最常见病原体。
传统的STUI病原学诊断实验室技术条件要求高。因此，建立一种能够针对引起STUI的多种常见病原体同时快速检测的方法，并用于指导临床的诊治很有必要。本研究旨在利用基因芯片技术，结合通用引物PCR(UPPCR)和多重PCR(MPCR)技术，建立同时快速检测多种引起STUI常见病原体的方法。
第一章：检测STUI常见病原体三对通用引物的设计、筛选及靶基因的确定。
首先将待检测的9种目的病原体分为三类：病毒类(HSV-1、HSV-2)、细菌类(淋病双球菌、解脲脲原体、沙眼衣原体、人型支原体、生殖支原体)及低级真核生物类(白念珠菌、阴道毛滴虫)。在美国生物信息中心(NCBI)的GenBank数据库中获得目的基因(单纯疱疹病毒1、2型的DNA聚合酶基因，细菌类的16S rRNA基因，低级真核生物类的18S rRNA基因)的核苷酸序列，并进行了BLASTn查询分析。利用专业软件Primer Premier 5.0和Oligo 6.67针对上述各目的基因，设计和筛选了三类通用引物，并了解了这些引物的所有动力学特性(包括引物的长度、位置、PCR产物的长度、位置、Tm值、二级结构、上下游引物的错配、3’端的稳定性及GC含量等)。再用多重PCR技术，针对上述三类通用引物进行设计，选择动力学性质尽可能相近的引物对，并进行不同引物及引物与产物之间的模拟杂交分析，优化引物在同一反应体系时的反应参数。最终筛选出分别针对病毒类、细菌类、低级真核生物类的三对通用引物。获得了不同引物对相应的靶基因在GenBank数据库的位置和序列。
第二章：检测STUI常见病原体三对通用引物实验室验证及复合PCR的建立
An increasing prevalence of sexually transmitted urogenital infection (STUI) over the recent decades has become a profound effect on the health of human beings, especially of nongonococcal urethritis (NGU), because of its various kinds of pathogens, latent infection, and asymptomatic pathogen shedding.
many studies have confirmed that STUI is commonly caused by the pathogens such as: Neisseria gonorrhoeae(NG), Trichomonas vaginalis(T. vaginalis), Chlamydia trachomatis(CT), Herpes simplex virus(HSV-1, HSV-2), Ureaplasma urealyticum(UU), Mycoplasma genitalium(MG), Mycoplasma hominis(MH), Candida albicans(C. albicans).
The conventional methodologies, such as direct smear, bacterial or viral culture and immunoassay, are not only time-consuming and laborious but also lowly sensitive and accurate, have been continuously applied for detecting pathogens causing sexually transmitted urogenital infection (STUI) for many years as a result of absence of other substituted methods. Clinically and epidemiologically, it is necessary to develop a simple method with higher sensitivity in order to improve clinical diagnosis and epidemiological surveys. In view of this and through a systematic approach, i.e. reviewing relevant information/literatures, by combining with universal primer PCR and multiplex PCR techniques, STUI gene chip technique was developed to enable a single test to simultaneously detect various pathogens with a high specificity, sensitivity and accuracy.
Chapter One: Design and Screening of Universal Primers, and Identification of Target Genes. First, we devided the target pathogens into three types: bacteria(Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma genitalium, Mycoplasma hominis),viruses(HSV-1, HSV-2) and low eukaryotes( Trichomonas vaginalis, Candida albicans), Follwing the relevant references, the genes located at conservative regions of 9 pathogens, including DNA
研究已证实淋病双球菌、解脲脲原体、沙眼衣原体、人型支原体、生殖支原体、单纯疱疹病毒Ⅰ(HSV-1)、单纯疱疹病毒Ⅱ(HSV-2)、白念珠菌及阴道毛滴虫为引起性传播泌尿生殖道感染(Sexually Transmitted Urogenital Infections，STUI)的最常见病原体。
传统的STUI病原学诊断实验室技术条件要求高。因此，建立一种能够针对引起STUI的多种常见病原体同时快速检测的方法，并用于指导临床的诊治很有必要。本研究旨在利用基因芯片技术，结合通用引物PCR(UPPCR)和多重PCR(MPCR)技术，建立同时快速检测多种引起STUI常见病原体的方法。
第一章：检测STUI常见病原体三对通用引物的设计、筛选及靶基因的确定。
首先将待检测的9种目的病原体分为三类：病毒类(HSV-1、HSV-2)、细菌类(淋病双球菌、解脲脲原体、沙眼衣原体、人型支原体、生殖支原体)及低级真核生物类(白念珠菌、阴道毛滴虫)。在美国生物信息中心(NCBI)的GenBank数据库中获得目的基因(单纯疱疹病毒1、2型的DNA聚合酶基因，细菌类的16S rRNA基因，低级真核生物类的18S rRNA基因)的核苷酸序列，并进行了BLASTn查询分析。利用专业软件Primer Premier 5.0和Oligo 6.67针对上述各目的基因，设计和筛选了三类通用引物，并了解了这些引物的所有动力学特性(包括引物的长度、位置、PCR产物的长度、位置、Tm值、二级结构、上下游引物的错配、3’端的稳定性及GC含量等)。再用多重PCR技术，针对上述三类通用引物进行设计，选择动力学性质尽可能相近的引物对，并进行不同引物及引物与产物之间的模拟杂交分析，优化引物在同一反应体系时的反应参数。最终筛选出分别针对病毒类、细菌类、低级真核生物类的三对通用引物。获得了不同引物对相应的靶基因在GenBank数据库的位置和序列。
第二章：检测STUI常见病原体三对通用引物实验室验证及复合PCR的建立
An increasing prevalence of sexually transmitted urogenital infection (STUI) over the recent decades has become a profound effect on the health of human beings, especially of nongonococcal urethritis (NGU), because of its various kinds of pathogens, latent infection, and asymptomatic pathogen shedding.
many studies have confirmed that STUI is commonly caused by the pathogens such as: Neisseria gonorrhoeae(NG), Trichomonas vaginalis(T. vaginalis), Chlamydia trachomatis(CT), Herpes simplex virus(HSV-1, HSV-2), Ureaplasma urealyticum(UU), Mycoplasma genitalium(MG), Mycoplasma hominis(MH), Candida albicans(C. albicans).
The conventional methodologies, such as direct smear, bacterial or viral culture and immunoassay, are not only time-consuming and laborious but also lowly sensitive and accurate, have been continuously applied for detecting pathogens causing sexually transmitted urogenital infection (STUI) for many years as a result of absence of other substituted methods. Clinically and epidemiologically, it is necessary to develop a simple method with higher sensitivity in order to improve clinical diagnosis and epidemiological surveys. In view of this and through a systematic approach, i.e. reviewing relevant information/literatures, by combining with universal primer PCR and multiplex PCR techniques, STUI gene chip technique was developed to enable a single test to simultaneously detect various pathogens with a high specificity, sensitivity and accuracy.
Chapter One: Design and Screening of Universal Primers, and Identification of Target Genes. First, we devided the target pathogens into three types: bacteria(Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma genitalium, Mycoplasma hominis),viruses(HSV-1, HSV-2) and low eukaryotes( Trichomonas vaginalis, Candida albicans), Follwing the relevant references, the genes located at conservative regions of 9 pathogens, including DNA
引文
1．徐文严主编．性传播疾病预防与治疗．上海：第二军医大学出版社，2001
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5. Hamels S, Gala JL, Dufour S,et al. Consensus PCR and microarray for diagnosis of the genus??Staphylococcus, species, and methicillin resistance. Biotechniques,2001,31(6): 1364-1366, 1368, 1370-1372.
6. Head IM, Saunder JR, Pickup RW. Microbial evolution, diversity and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol, 1998,35:1～21.
7．刘爱英，孙建方，尹跃平，等．基因芯片技术检测生殖器溃疡性性病病原体．中华皮肤科杂志，2004，5：265-267．
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4. Head IM, Saunder JR, Pickup RW. Microbial evolution, diversity and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol, 1998,35:1～21.
5．刘爱英，孙建方，尹跃平，等．基因芯片技术检测生殖器溃疡性性病病原体．中华皮肤科杂志，2004，5：265-267．1. Harris KA, Hartley JC. Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol,2003,52(Pt 8):685-691.
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9. Loy A, Horn M, Wagner M. probeBase: an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res, 2003,31(1):514-516.
10. Kaydos SC, Swygard H, Wise SL, et al. Development and validation of a PCR-based enzyme-linked im munosorbent assay with urine for use in clinical research settings to detect Trichomonas vaginalis in women. J Clin Microbiol, 2002,40(1):89-95.
11. Aho J, Kornegay JR, Healey S, et al. Evaluation of a convenient enzyme immunoassay to assess the quality of genital specimens submitted for the detection of human papillomavirus DNA by consensus PCR. J Clin Virol, 2004,29(2):127-133.
12. Lu JJ, Perng CL, Lee SY, et al. Use of PCR with universal primers and restriction endonuclease digestions for detection and identification of common bacterial pathogens in cerebrospinal fluid. J Clin Microbiol, 2000,38(6):2076-2080.
13. Sakulwira K, Vanapongtipagorn P, Theamboonlers A, et al. Detection and differentiation of human herpesviruses 1-5 by consensus primer PCR and RFLP. Asian Pac J Allergy Immunol, 2003,21(1):55-61.
14. Gingeras TR, Ghandour G, Wang E, et al. Simultaneous genotyping and species identification using hybridization pattern recognition analysis of generic Mycobacterium DNA arrays. Genome Res, 1998, 8(5):435-448.
15. Chandler DP, Newton GJ, Small JA, et al. Daly DS. Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays. Appl Environ Microbiol, 2003,69(5):2950-2958.
16. Anthony RM, Brown TJ, French GL. Rapid diagnosis of bacteremia by universal amplification of??23S ribosomal DNA followed by hybridization to an oligonucleotide array. J Clin Microbiol, 2000,38(2):781-788.
17. Johnson G, Nelson S, Petric M, et al. Comprehensive PCR-based assay for detection and species identification of human herpesviruses. J Clin Microbiol, 2000,38(9):3274-3279.
18. Ferrero DV, Meyers HN, Schultz DE, et al. Performance of the Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay in detecting Chlamydia trachomatis in endocervical and urine specimens from women and urethral and urine specimens from men attending sexually transmitted disease and family planning clinics. J Clin Microbiol, 1998,36(11):3230-3233.1．刘爱英，孙建方，尹跃平，等．基因芯片技术检测生殖器溃疡性性病病原体．中华皮肤科杂志，2004，5：265．267．
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6. Head IM, Saunder JR, Pickup RW. Microbial evolution, diversity and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol, 1998,35:1～21.
7．刘爱英，孙建方，尹跃平，等．基因芯片技术检测生殖器溃疡性性病病原体．中华皮肤科杂志，2004，5：265-267．
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9. Loy A, Horn M, Wagner M. probeBase: an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res, 2003,31(1):514-516.
10. Kaydos SC, Swygard H, Wise SL, et al. Development and validation of a PCR-based enzyme-linked im munosorbent assay with urine for use in clinical research settings to detect Trichomonas vaginalis in women. J Clin Microbiol, 2002,40(1):89-95.
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16. Anthony RM, Brown TJ, French GL. Rapid diagnosis of bacteremia by universal amplification of??23S ribosomal DNA followed by hybridization to an oligonucleotide array. J Clin Microbiol, 2000,38(2):781-788.
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