沙眼衣原体高分辨率溶解曲线基因分型方法的建立及其在男男性行为者流行病学调查中的应用
发布时间：2018-07-17 02:26
【摘要】：沙眼衣原体(Chlamydia trachomatis, CT)分15种血清型,其中A-C型是引起沙眼的主要病原体,D-K型主要引起泌尿生殖道感染,L1-L3型可以经性接触传播发生系统性感染而导致性病性淋巴肉芽肿(Lymphogranuloma Venereum, LGV)。大部分的CT感染没有临床症状,但感染可持续存在并引起一系列女性、男性和新生儿的临床疾病和后遗症。LGV累及生殖器、肛门、直肠及淋巴结等,导致溃疡、脓肿和瘘管的发生,晚期可引起外生殖器橡皮肿、挛缩性瘢痕和直肠狭窄等严重后遗症。 CT是目前世界范围内引起性传播感染的最常见的病原体,据世界卫生组织估计,每年全球新感染的CT感染病例数约为9200万例。CT感染在许多发达国家已经超过淋病居所有STI的首位。LGV常见于热带和亚热带地区,但自2003年底来在欧洲和北美等国家的男男性行为人群(Men who have sex with men,MSM)中发生了爆发流行。我国90年代后报道的LGV疑似病例大多由于缺乏有效地实验室诊断手段未得到确诊。 目前的CT基因分型技术已经由建立在主要外膜蛋白(Major outer membrane protein, MOMP)单克隆抗体基础上的血清学分型发展到建立在CT的ompA基因扩增基础上的基因分型。但这些分型方法都需额外步骤的PCR后分析,操作繁琐、费时和容易污染,不适于大规模的流行病学调查和LGV的临床诊断。实时荧光PCR技术(RT-PCR)已经应用到了CT基因分型研究,但主要是针对LGV型别,而且Taqman探针的价格高,易分解,限制了其在流行病学中的应用。 CT的基因分型研究有助于更好的了解不同基因型别的流行动态,地区和人群分布特征；有利于性传播网络的研究；了解感染和并发症发生的机制；同时为CT疫苗的研究奠定基础；CT基因分型还可作为诊断LGV的重要实验室诊断依据。MSM人群中CT感染的分子流行病学研究目前我国尚未见报道,此人群中LGV的流行情况尚缺乏基线资料。本研究的目的是建立一种高效、快捷、稳定的CT基因分型方法,并应用于MSM人群中CT感染的分子流行病学研究。 第一部分沙眼衣原体ompA基因序列的分析和目的扩增片段的确定 OmpA基因在CT内是单拷贝的,主要编码MOMP蛋白的氨基酸序列,总长度约1.1kb左右,包括4个可变区(variable segment, VS)和5个恒定区(constant segment, CS),其中VS区基因序列编码MOMP主要抗原决定簇的氨基酸序列,是CT基因分型的基础。为了筛选适合高分辨率溶解曲线基因分型方法(High-resolution melting analysis, HRMA)的VS区域,我们从Genbank获得15种型别的CT的ompA基因序列,用DNAstar5.0生物信息学软件对ompA基因和4个VS区基因序列进行分析,初步确定片段最短,各型间离散性最大的VS2区作为HRMA基因分型的靶分析区域；然后在对15个型别的CT标准菌株进行复苏、传代培养的基础上,设计引物扩增ompA基因VS1-VS2区,进一步分析VS区基因序列；最后通过对36株临床阳性标本的ompA基因VS1-VS2区扩增和变异性分析,同时结合文献检索的结果,最终确定VS2区作为HRMA基因分型的扩增和分析片段。 第二部分沙眼衣原体高分辨率溶解曲线基因分型方法的建立 HRMA是一种全新的DNA扩增后分析技术,配合使用渗透性饱和染料,PCR扩增后升高温度并监测DNA双链解开过程中荧光强度的变化,得到溶解曲线从而实现对PCR扩增产物的分析。我们设计了针对ompA基因VS2区的通用巢氏RT-PCR扩增引物,扩增出11种与性传播感染相关的CT的ompA基因VS2区并运用HRMA方法进行基因分型,敏感性可达到每个反应1个拷贝,与10种泌尿生殖道常见的病原体和共生菌之间无交叉反应；临床标本检测显示其可对36份质粒扩增阳性的临床标本全部扩增并分型,分型结果与测序分型结果一致；创建了11种与性传播感染相关的沙眼衣原体的HRMA基因分型的工作流程。与现有的其他基因分型方法相比较,HRMA基因分型方法可在扩增后直接通过软件的溶解曲线分析和基因扫描分析来判断标本的基因型,而不需要繁琐的和额外步骤PCR后处理分析,成本效果性好、快速、简单,并且大大减少了污染的几率；LCGreen饱和染料不仅性质稳定,易于储存,而且对DNA双链分子没有破坏性,经过HRMA分析后的扩增产物仍可进行琼脂糖凝胶电泳、测序等后续DNA分析。 第三部分男男性行为人群中沙眼衣原体的分子流行病学研究 我们以南部沿海的深圳市的MSM人群作为研究对象,2008年9月至2009年5月对深圳两家MSM桑拿浴场的145名MSM进行调查,其中有143人(98.6%)同意参加本研究,由外展工作人员进行问卷调查并现场采集直肠和尿液标本。直肠拭子和尿液标本进行CT和淋球菌检测,并对CT阳性标本进行基因分型。结果显示人群中直肠CT感染率为24.4%,尿道CT和淋球菌感染率分别为5.3%和1.5%,其中2.5%的MSM直肠和尿道同时感染CT,没有检测到CT和淋球菌同时感染的情况；单因素分析结果显示直肠炎症状与直肠CT感染之间的关联性具有统计学意义；基因分型结果显示直肠CT感染最常见的基因型别是G型,占所有阳性直肠标本的40%；其次是D型和J型,占36.6%和10%；E型和I型分别占3.3%和6.6%；检测到2例B型感染,占6.6%；没有检测到LGV型。统计分析结果显示深圳MSM人群中CT的基因型分布与中国其他异性恋人群中的CT基因型分布具有统计学意义的差别；同大多数国外的MSM人群中CT的基因型分布基本相同。
[Abstract]:Chlamydia trachoma ( CT ) is divided into 15 serotypes , of which A - C is the main pathogen causing trachoma , and the D - K type mainly causes urinary genital tract infection , and the L1 - L3 type can cause systemic infection through sexual contact and lead to sexually transmitted lymphogranuloma ( LGV ) . Most CT infections have no clinical symptoms , but the infection can persist and cause a series of clinical diseases and sequelae of female , male and newborn babies . LGV is involved in genital , anal , rectal and lymph nodes , leading to the occurrence of ulcers , abscess and fistula , which may cause severe sequelae such as rubber swelling , contracture scar , and rectal stenosis .
CT is the most common cause of sexually transmitted infections worldwide . According to the World Health Organization , the number of CT infections worldwide has been estimated to be approximately 92 million per year . CT infections have been in the forefront of all STI in many developed countries . LGV is common in tropical and subtropical regions , but most of the LGV suspected cases reported since the end of 2003 have not been diagnosed due to the lack of effective laboratory diagnostic tools .
At present , CT genotyping techniques have been developed by serological typing based on the monoclonal antibody of major outer membrane protein ( MOMP ) to establish genotyping on the basis of ompA gene amplification of CT . However , these typing methods require additional steps of PCR analysis , tedious operation , time consuming and easy contamination , and are not suitable for large - scale epidemiological investigation and clinical diagnosis of LGV . Real - time fluorescence PCR ( RT - PCR ) has been applied to the clinical diagnosis of LGV .
The genotyping of CT helps to better understand the epidemic dynamics , regional and population distribution characteristics of different genotypes .
It is beneficial to the research of sexually transmitted network ;
To understand the mechanism of infection and complication ;
and lays a foundation for the research of CT vaccine ;
CT gene typing can also be used as an important diagnostic basis for diagnosis of LGV . The molecular epidemiology of CT infection in MSM population is not reported in China . The prevalence of LGV in this population is still lack of baseline data . The aim of this study is to establish an efficient , fast and stable CT genotyping method and to apply the molecular epidemiology of CT infection in MSM population .
Analysis of the Sequence of ompA Gene of Chlamydia Tracts in the First Part and Determination of the Target Amplifying Fragment
OmpA鍩哄洜鍦–T鍐呮槸鍗曟嫹璐濈殑,涓昏缂栫爜MOMP铔嬬櫧鐨勬皑鍩洪吀搴忓垪,鎬婚暱搴︾害1.1kb宸﹀彸,鍖呮嫭4涓彲鍙樺尯(variable segment, VS)鍜，
本文编号：2128603
[Abstract]:Chlamydia trachoma ( CT ) is divided into 15 serotypes , of which A - C is the main pathogen causing trachoma , and the D - K type mainly causes urinary genital tract infection , and the L1 - L3 type can cause systemic infection through sexual contact and lead to sexually transmitted lymphogranuloma ( LGV ) . Most CT infections have no clinical symptoms , but the infection can persist and cause a series of clinical diseases and sequelae of female , male and newborn babies . LGV is involved in genital , anal , rectal and lymph nodes , leading to the occurrence of ulcers , abscess and fistula , which may cause severe sequelae such as rubber swelling , contracture scar , and rectal stenosis .
CT is the most common cause of sexually transmitted infections worldwide . According to the World Health Organization , the number of CT infections worldwide has been estimated to be approximately 92 million per year . CT infections have been in the forefront of all STI in many developed countries . LGV is common in tropical and subtropical regions , but most of the LGV suspected cases reported since the end of 2003 have not been diagnosed due to the lack of effective laboratory diagnostic tools .
At present , CT genotyping techniques have been developed by serological typing based on the monoclonal antibody of major outer membrane protein ( MOMP ) to establish genotyping on the basis of ompA gene amplification of CT . However , these typing methods require additional steps of PCR analysis , tedious operation , time consuming and easy contamination , and are not suitable for large - scale epidemiological investigation and clinical diagnosis of LGV . Real - time fluorescence PCR ( RT - PCR ) has been applied to the clinical diagnosis of LGV .
The genotyping of CT helps to better understand the epidemic dynamics , regional and population distribution characteristics of different genotypes .
It is beneficial to the research of sexually transmitted network ;
To understand the mechanism of infection and complication ;
and lays a foundation for the research of CT vaccine ;
CT gene typing can also be used as an important diagnostic basis for diagnosis of LGV . The molecular epidemiology of CT infection in MSM population is not reported in China . The prevalence of LGV in this population is still lack of baseline data . The aim of this study is to establish an efficient , fast and stable CT genotyping method and to apply the molecular epidemiology of CT infection in MSM population .
Analysis of the Sequence of ompA Gene of Chlamydia Tracts in the First Part and Determination of the Target Amplifying Fragment
OmpA鍩哄洜鍦–T鍐呮槸鍗曟嫹璐濈殑,涓昏缂栫爜MOMP铔嬬櫧鐨勬皑鍩洪吀搴忓垪,鎬婚暱搴︾害1.1kb宸﹀彸,鍖呮嫭4涓彲鍙樺尯(variable segment, VS)鍜，
本文编号：2128603
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