生殖支原体Taqman MGB荧光PCR方法的建立及在高危人群流行病学调查中的应用
发布时间：2018-07-26 11:14
【摘要】：生殖支原体(Mycoplasma genitalium, Mg)是近年来确认的性传播疾病(STD)病原体之一,在男性引起非淋球菌性尿道炎(NGU),在女性导致宫颈炎、子宫内膜炎及盆腔炎等,如不治疗可致异位妊娠、不孕等不良结局。 NUG是常见的STD,世界范围而言,每年有8900万的新发病例,NGU的发病率呈逐年上升的趋势。在我国,自1988年起,NGU作为重点监测的性病在一些监测点对其流行情况进行监测,2002年以来NGU的病例数超过淋病,居所有规定上报的STD首位,在随后几年中均占全国STD病例报告数构成比的35%以上,然而自2008年起NGU的监测被沙眼衣原体(CT)感染所取代,最主要的原因是CT感染已被确认为NGU的主要病因之一,可在11%-43%的NGU患者中检出。 研究表明Mg是NGU的另一个重要的常见病原之一,在9%--25%NGU患者中可检出,与CT合并感染少见,规范治疗后复发性及持续性NGU患者中Mg阳性多见,因此在CT阴性的NGU患者中Mg可能发挥重要作用。采用推荐治疗药物阿奇霉素或强力霉素规范治疗后,对Mg阳性的NGU治疗失败的病例陆续有报道,说明有耐药的产生。随着对Mg在NGU中致病作用的认识,Mg在国外日益得到重视,2009年的欧洲男性NGU诊疗指南中提到需要把Mg的检测纳入临床诊疗中,对慢性NGU患者的治疗应该覆盖治疗Mg的药物,2010年东欧国家确立了关于Mg感染的实验室诊断指南。 对Mg在中国人群中流行情况的研究比较有限,既往研究发现在NGU患者中Mg的流行率为20.6%-25%,在CT阴性的NGU患者中可达44.19%,此发现与其他一些国家的研究类似,说明Mg在中国的流行应引起重视。 女性性工作者(FSWs),性病门诊男性就诊者(MSP)及男男性行为者(MSM)是STD/HIV感染的高危人群,也是HIV哨点监测的重点人群,随着近年来HIV传播模式的转变,经性传播已成为HIV传播的主要模式,上述人群在HIV的传播起着重要作用。近来研究发现Mg与HIV密切相关,Mg感染可能促进HIV的感染及传播,一项对泌尿生殖道Mg感染与HIV阳性的相关性的系统性综述及Meta分析发现Mg感染与HIV阳性显著相关,Mg感染者中HIV阳性率是Mg未感染者的2倍,此外有研究报道Mg感染可能通过促进HIVDNA的脱落进而促进HIV的传播。因此了解上述高危人群中Mg的流行情况,及时干预,不仅有助于控制Mg的传播,预防不良结局的发生,而且可能对于HIV的防治也起着积极作用。 尽管Mg在国际社会越来越受到重视,然而由于Mg体外培养极其困难,且目前国内没有商品化检测试剂盒等,因此大大限制了对Mg的流行病学研究,缺乏相应的流行病学资料。本研究旨在采用文献报道的简便快捷,敏感性高,特异性好的TaqmanMGB实时定量PCR方法对MSP中Mg的流行情况进行调查,并对相关危险因素进行分析,为高危人群中Mg的流行及监测提供基线资料,为制定防治策略提供参考。全文分为三部分,各部分主要内容分述如下： 第一部分：Taqman MGB荧光PCR方法建立 采用文献中报道特异性及敏感性较高的以Mg主要黏附素基因(MgPa基因)为靶基因的Taqman MGB荧光PCR方法,用Mg标准菌株G37制备浓度分别为1×101拷贝/gl、1×102拷贝/gl、1×103拷贝/μl、1×104拷贝/μl、1×10。拷贝/μl、1×106拷贝/μl的6个标准品,制备标准曲线,并对该方法的重复性、灵敏度及特异性进行检测,确定该方法的检测限(LOD)及定量限(LOQ)。结果显示Taqman MGB实时定量PCR方法检测的线性范围好(1×101拷贝/μl--l×106拷贝/μl),标准曲线回归方程y=-3.982x+40.80,R2=0.993,重复性好(批内变异系数=0.7%,批间变异系数=1.09%),敏感性高,检测限(LOD)为每个PCR反应10拷贝,定量限(LOQ)为10拷贝/μl,用该方法检测泌尿生殖道常见10种病原体均为阴性,说明该方法灵敏度高,特异性好,且有较好的重复性。我们认为针对MgPa基因的Taqman MGB实时PCR可以快速、灵敏的对Mg进行定性及定量的检测,为检测人群中Mg的感染情况提供了极大的便利。 第二部分：广西女性性工作者及性病门诊男性就诊者中生殖支原体的流行病学调查及危险因素分析 采用第一部分建立的Taqman MGB荧光PCR方法对广西FSWs及MSP中Mg的流行情况进行调查,采用基于场所的分层抽样方法,于2009年7月至9月对广西梧州、贺州的宾馆、酒店、夜总会、KTV、发廊、路边店、站街、公园等娱乐场所及室外场所的FSWs及贺州性病门诊就诊的男性进行调查。按工作场所及每次性交易收入将FSWs划分为3类,路边店、出租屋、站街及公园工作或每次性交易低于50元者为低档FSWs, KTV、发廊、美容美发等场所工作者为中档FSWs,宾馆、酒店、夜总会为高档FSWs,取得知情同意后对受试者进行问卷调查并采集FSWs的宫颈拭子及MSP的尿液标本,Taqman MGB荧光PCR检测Mg。结果共纳入810名FSWs及423名MSP,收集到810份宫颈拭子及407份尿液标本。FSWs中Mg的感染率为13.2%(95%CI=10.87%-15.52%),高中低档FSWs Mg感染率分别为15.7%(95%CI=13.12%-18.2%),10.8%(95%CI=8.67%-12.93%)及14.6%(95%CI=12.17%-17.03%),不同档次间无统计学差异,受教育程度低于初中(AOR=2.36,95%CI=1.15-4.87),未婚(AOR=2.27,95%CI=1.42-3.62),非当地居民(AOR=2.03,95%CI=1.29-3.20),过去1年无STIs相关症状(AOR=1.66,95%CI=1.09-2.52)与Mg感染显著相关。MSP中Mg感染率为28%(95%CI=25.50%-30.49%),独居(AOR=3.84,95%CI=1.58-9.33),过去1年无STIs相关症状(AOR=2.21,95%CI=1.35-3.62),最近3个月与FSWs发生性行为(AOR=2.27,95%CI=1.41-3.67)与Mg感染相关。以上研究结果表明在FSWs及MSP人群中Mg感染率高,且多为无症状感染,提示对上述高危人群中Mg感染进行常规筛查成为必要。 第三部分：广州男男性行为者中生殖支原体的流行率调查及危险因素分析 于2010年1月至5月采用滚雪球的方法对广州市同性恋酒吧、茶吧、桑拿浴池、夜总会、公园、公厕等场所的MSM进行问卷调查并采集尿液标本,Taqman MGB荧光PCR检测Mg。328名MSM中,97.6%自我认同为同性恋,其中41.8%虽自我认同为同性恋但也与女性发生性关系,87.8%半年内与男性发生过肛交,63.2%最近一次肛交中使用了安全套,16.8%半年内与女性发生过性行为,仅27.3%在最近一次与女性发生性行为时使用了安全套,4.6%参加过性交易,寻找性伴的方式以互联网为主,占79.0%。323份尿液标本中Mg感染率为4.3%(95%CI=3.1%-5.4%),未发现与Mg感染相关的因素。研究结果表明在MSM人群中Mg感染率低,提示Mg感染以异性性行为途径传播为主,MSM作为Mg感染的传染源意义更大。
[Abstract]:Mycoplasma genitalium (Mg) is one of the pathogens of sexually transmitted disease (STD) identified in recent years. It causes non gonococcal urethritis (NGU) in men, causes cervicitis, endometritis and pelvic inflammation in women, such as untreated undesirable outcomes, such as ectopic pregnancy and infertility.
NUG is a common STD. In the world, there are 89 million new cases each year, and the incidence of NGU is increasing year by year. In China, since 1988, NGU has been monitored as a key monitoring venereal disease at some monitoring points. Since 2002, the number of cases of NGU exceeds the gonorrhea, and there is a set of STD first reported in the residence, and then in the following. For a few years, more than 35% of the national STD case reports were accounted for. However, since 2008, NGU monitoring was replaced by Chlamydia trachomatis (CT) infection. The most important reason is that CT infection has been identified as one of the main causes of NGU, and can be detected in NGU patients in 11%-43%.
The study shows that Mg is one of the other important common pathogens of NGU, which can be detected in 9%--25%NGU patients. It is rare to merge with CT, and Mg positive in recurrent and persistent NGU patients after standard treatment. Therefore, Mg may play an important role in the CT negative NGU patients. The recommended treatment for azithromycin or doxycycline is a standard treatment. After treatment, the cases of Mg positive NGU failure have been reported in succession, indicating the emergence of drug resistance. With the understanding of the pathogenicity of Mg in NGU, Mg is becoming more and more important abroad. In 2009, the European male NGU guide to diagnosis of Mg should be included in the clinical diagnosis of Mg, and the treatment of chronic NGU patients should be covered with the drugs for the treatment of Mg. In 2010, Eastern European countries established guidelines for laboratory diagnosis of Mg infection.
Research on the prevalence of Mg in Chinese population is limited. Previous studies have found that the prevalence of Mg in NGU patients is 20.6%-25% and is up to 44.19% in CT negative NGU patients. This discovery is similar to that in other countries, indicating that the prevalence of Mg in China should be paid more attention.
Female sex workers (FSWs), STD clinic male doctor (MSP) and male male actor (MSM) are high risk population of STD/HIV infection, and also the key population of HIV sentinel monitoring. With the change of HIV transmission pattern in recent years, sexual transmission has become the main mode of HIV transmission. The spread of the above population plays an important role in the transmission of HIV. Recently, the research has been studied. Mg is closely related to HIV. Mg infection may promote infection and transmission of HIV. A systematic review of the correlation between Mg infection and HIV positive in genitourinary tract and Meta analysis found that Mg infection is significantly related to HIV positive. The HIV positive rate in Mg infected persons is 2 times that of those who are not infected with Mg. Falling off further promotes the spread of HIV. Therefore, understanding the prevalence of Mg in these high-risk groups and intervening in time will not only help to control the spread of Mg, prevent the occurrence of bad outcome, but also play an active role in the prevention and control of HIV.
Although Mg is being paid more and more attention in the international community, however, it is very difficult to cultivate Mg in vitro, and there is no commercial detection kit in China at present. Therefore, the epidemiological study of Mg is greatly restricted and the corresponding epidemiological data are lacking. This study aims to adopt the simple, fast, sensitive and specific TaqmanM of the literature report. GB real-time quantitative PCR method was used to investigate the prevalence of Mg in MSP, and the related risk factors were analyzed to provide baseline data for the epidemic and monitoring of Mg in high-risk groups, and to provide reference for the development of prevention and control strategies. The full text is divided into three parts, the main contents of each part are as follows:
The first part: the establishment of Taqman MGB fluorescence PCR method
The Taqman MGB fluorescent PCR method, which was reported in the literature, was specific and sensitive to Mg major adhesion gene (MgPa gene) as the target gene. The Mg standard strain G37 was used to prepare 1 x 101 copies /gl, 1 x 102 copies /gl, 1 x 103 copies / mu L, 1 * 104 copy / mu L, 1 x 10. copy / muon, 1 106 copy / micron standard, preparation standard, preparation standard Curves, and detection of the repeatability, sensitivity and specificity of the method, determine the detection limit (LOD) and quantitative limit (LOQ) of the method. The results show that the linear range of the Taqman MGB real-time quantitative PCR method is good (1 x 101 copies / mu l--l x 106 copies / L), the standard curve regression equation y=-3.982x+40.80, R2=0.993, and good reproducibility (intra variation) The coefficient =0.7%, the inter group variation coefficient =1.09%), the sensitivity high, the detection limit (LOD) for each PCR reaction 10 copies, the quantitative limit (LOQ) is 10 copies / mu L, using this method to detect the genitourinary tract common 10 pathogens are negative, indicating that the method is high sensitivity, good specificity, and has good repeatability. We think of the MgPa gene Taqman MGB real. PCR can quickly and sensitively detect the Mg qualitatively and quantitatively. It provides a great convenience for detecting the infection of Mg in the population.
The second part: epidemiological investigation and risk factors analysis of Mycoplasma genital mycoplasma in male sex workers and sexually transmitted diseases outpatients in Guangxi.
The Taqman MGB fluorescence PCR method established in the first part was used to investigate the epidemic situation of Mg in Guangxi FSWs and MSP. By using the place based stratified sampling method, the FSWs and Hezhou sex of the hotels, hotels, nightclubs, KTV, hairdresser, roadside, park, park and other places of entertainment and outdoor places in Wuzhou, Wuzhou, Hezhou, Hezhou, Guangxi, from July 2009 to September. FSWs is divided into 3 categories according to the workplace and the income of each sex transaction. Roadside stores, rental houses, station street and park work or lower FSWs, KTV, hairdresser, beauty hairdresser and other place workers are low file FSWs, KTV, hairdresser, and beauty hairdresser for the middle grade FSWs, hotel, hotel and night club for the high-end FSWs, after obtaining informed consent The subjects were investigated with a questionnaire and collected FSWs's cervix swabs and MSP urine specimens. The results of Taqman MGB fluorescence PCR were included in 810 FSWs and 423 MSP. The infection rate of Mg in 810 cervix swabs and 407 urine specimens was 13.2% (95%CI=10.87%-15.52%) and 15.7% (15.7%) 2%-18.2%), 10.8% (95%CI=8.67%-12.93%) and 14.6% (95%CI=12.17%-17.03%), there was no statistical difference between different grades, the degree of education was lower than that of junior middle school (AOR=2.36,95%CI=1.15-4.87), unmarried (AOR=2.27,95%CI=1.42-3.62), non local residents (AOR=2.03,95%CI= 1.29-3.20), and no STIs related symptoms (AOR=1.66,95%CI=1.09-2.52) and Mg infection had been found in the past 1 years. The incidence of Mg infection in the associated.MSP was 28% (95%CI=25.50%-30.49%), solitary (AOR=3.84,95%CI=1.58-9.33), without STIs related symptoms (AOR=2.21,95%CI=1.35-3.62) in the last 1 years, and the recent 3 months with FSWs sexual behavior (AOR=2.27,95%CI=1.41-3.67) associated with Mg infection. The above study results showed that the Mg rate was high in FSWs and MSP population, and most of them were asymptomatic. It is suggested that routine screening for Mg infection in these high-risk groups is necessary.
The third part: prevalence and risk factors of Mycoplasma reproductive tract among men who have sex with men in Guangzhou.
From January 2010 to May, the method of snowball was used to investigate and collect urine samples from MSM gay bar, tea bar, sauna bath pool, night club, park, public toilet and other places. Taqman MGB fluorescence PCR was used to detect Mg.328 name MSM, 97.6% self identity was homosexual, of which 41.8% were homosexual but also female hair. Sexual relationship, 87.8% half a year had anal sex with men, 63.2% in the last time in the use of condoms in the anal intercourse, 16.8% months with women in sexual behavior, only 27.3% in the last time with women in the use of condoms, 4.6% sex trade, the way to find sex with the Internet, accounting for 79.0%.323 urine specimens. The infection rate of Mg was 4.3% (95%CI=3.1%-5.4%), and the factors associated with Mg infection were not found. The results showed that the rate of Mg infection was low in the MSM population, suggesting that Mg infection was mainly transmitted by heterosexual behavioral pathways, and MSM was more significant as the source of Mg infection.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R759
本文编号：2145830
[Abstract]:Mycoplasma genitalium (Mg) is one of the pathogens of sexually transmitted disease (STD) identified in recent years. It causes non gonococcal urethritis (NGU) in men, causes cervicitis, endometritis and pelvic inflammation in women, such as untreated undesirable outcomes, such as ectopic pregnancy and infertility.
NUG is a common STD. In the world, there are 89 million new cases each year, and the incidence of NGU is increasing year by year. In China, since 1988, NGU has been monitored as a key monitoring venereal disease at some monitoring points. Since 2002, the number of cases of NGU exceeds the gonorrhea, and there is a set of STD first reported in the residence, and then in the following. For a few years, more than 35% of the national STD case reports were accounted for. However, since 2008, NGU monitoring was replaced by Chlamydia trachomatis (CT) infection. The most important reason is that CT infection has been identified as one of the main causes of NGU, and can be detected in NGU patients in 11%-43%.
The study shows that Mg is one of the other important common pathogens of NGU, which can be detected in 9%--25%NGU patients. It is rare to merge with CT, and Mg positive in recurrent and persistent NGU patients after standard treatment. Therefore, Mg may play an important role in the CT negative NGU patients. The recommended treatment for azithromycin or doxycycline is a standard treatment. After treatment, the cases of Mg positive NGU failure have been reported in succession, indicating the emergence of drug resistance. With the understanding of the pathogenicity of Mg in NGU, Mg is becoming more and more important abroad. In 2009, the European male NGU guide to diagnosis of Mg should be included in the clinical diagnosis of Mg, and the treatment of chronic NGU patients should be covered with the drugs for the treatment of Mg. In 2010, Eastern European countries established guidelines for laboratory diagnosis of Mg infection.
Research on the prevalence of Mg in Chinese population is limited. Previous studies have found that the prevalence of Mg in NGU patients is 20.6%-25% and is up to 44.19% in CT negative NGU patients. This discovery is similar to that in other countries, indicating that the prevalence of Mg in China should be paid more attention.
Female sex workers (FSWs), STD clinic male doctor (MSP) and male male actor (MSM) are high risk population of STD/HIV infection, and also the key population of HIV sentinel monitoring. With the change of HIV transmission pattern in recent years, sexual transmission has become the main mode of HIV transmission. The spread of the above population plays an important role in the transmission of HIV. Recently, the research has been studied. Mg is closely related to HIV. Mg infection may promote infection and transmission of HIV. A systematic review of the correlation between Mg infection and HIV positive in genitourinary tract and Meta analysis found that Mg infection is significantly related to HIV positive. The HIV positive rate in Mg infected persons is 2 times that of those who are not infected with Mg. Falling off further promotes the spread of HIV. Therefore, understanding the prevalence of Mg in these high-risk groups and intervening in time will not only help to control the spread of Mg, prevent the occurrence of bad outcome, but also play an active role in the prevention and control of HIV.
Although Mg is being paid more and more attention in the international community, however, it is very difficult to cultivate Mg in vitro, and there is no commercial detection kit in China at present. Therefore, the epidemiological study of Mg is greatly restricted and the corresponding epidemiological data are lacking. This study aims to adopt the simple, fast, sensitive and specific TaqmanM of the literature report. GB real-time quantitative PCR method was used to investigate the prevalence of Mg in MSP, and the related risk factors were analyzed to provide baseline data for the epidemic and monitoring of Mg in high-risk groups, and to provide reference for the development of prevention and control strategies. The full text is divided into three parts, the main contents of each part are as follows:
The first part: the establishment of Taqman MGB fluorescence PCR method
The Taqman MGB fluorescent PCR method, which was reported in the literature, was specific and sensitive to Mg major adhesion gene (MgPa gene) as the target gene. The Mg standard strain G37 was used to prepare 1 x 101 copies /gl, 1 x 102 copies /gl, 1 x 103 copies / mu L, 1 * 104 copy / mu L, 1 x 10. copy / muon, 1 106 copy / micron standard, preparation standard, preparation standard Curves, and detection of the repeatability, sensitivity and specificity of the method, determine the detection limit (LOD) and quantitative limit (LOQ) of the method. The results show that the linear range of the Taqman MGB real-time quantitative PCR method is good (1 x 101 copies / mu l--l x 106 copies / L), the standard curve regression equation y=-3.982x+40.80, R2=0.993, and good reproducibility (intra variation) The coefficient =0.7%, the inter group variation coefficient =1.09%), the sensitivity high, the detection limit (LOD) for each PCR reaction 10 copies, the quantitative limit (LOQ) is 10 copies / mu L, using this method to detect the genitourinary tract common 10 pathogens are negative, indicating that the method is high sensitivity, good specificity, and has good repeatability. We think of the MgPa gene Taqman MGB real. PCR can quickly and sensitively detect the Mg qualitatively and quantitatively. It provides a great convenience for detecting the infection of Mg in the population.
The second part: epidemiological investigation and risk factors analysis of Mycoplasma genital mycoplasma in male sex workers and sexually transmitted diseases outpatients in Guangxi.
The Taqman MGB fluorescence PCR method established in the first part was used to investigate the epidemic situation of Mg in Guangxi FSWs and MSP. By using the place based stratified sampling method, the FSWs and Hezhou sex of the hotels, hotels, nightclubs, KTV, hairdresser, roadside, park, park and other places of entertainment and outdoor places in Wuzhou, Wuzhou, Hezhou, Hezhou, Guangxi, from July 2009 to September. FSWs is divided into 3 categories according to the workplace and the income of each sex transaction. Roadside stores, rental houses, station street and park work or lower FSWs, KTV, hairdresser, beauty hairdresser and other place workers are low file FSWs, KTV, hairdresser, and beauty hairdresser for the middle grade FSWs, hotel, hotel and night club for the high-end FSWs, after obtaining informed consent The subjects were investigated with a questionnaire and collected FSWs's cervix swabs and MSP urine specimens. The results of Taqman MGB fluorescence PCR were included in 810 FSWs and 423 MSP. The infection rate of Mg in 810 cervix swabs and 407 urine specimens was 13.2% (95%CI=10.87%-15.52%) and 15.7% (15.7%) 2%-18.2%), 10.8% (95%CI=8.67%-12.93%) and 14.6% (95%CI=12.17%-17.03%), there was no statistical difference between different grades, the degree of education was lower than that of junior middle school (AOR=2.36,95%CI=1.15-4.87), unmarried (AOR=2.27,95%CI=1.42-3.62), non local residents (AOR=2.03,95%CI= 1.29-3.20), and no STIs related symptoms (AOR=1.66,95%CI=1.09-2.52) and Mg infection had been found in the past 1 years. The incidence of Mg infection in the associated.MSP was 28% (95%CI=25.50%-30.49%), solitary (AOR=3.84,95%CI=1.58-9.33), without STIs related symptoms (AOR=2.21,95%CI=1.35-3.62) in the last 1 years, and the recent 3 months with FSWs sexual behavior (AOR=2.27,95%CI=1.41-3.67) associated with Mg infection. The above study results showed that the Mg rate was high in FSWs and MSP population, and most of them were asymptomatic. It is suggested that routine screening for Mg infection in these high-risk groups is necessary.
The third part: prevalence and risk factors of Mycoplasma reproductive tract among men who have sex with men in Guangzhou.
From January 2010 to May, the method of snowball was used to investigate and collect urine samples from MSM gay bar, tea bar, sauna bath pool, night club, park, public toilet and other places. Taqman MGB fluorescence PCR was used to detect Mg.328 name MSM, 97.6% self identity was homosexual, of which 41.8% were homosexual but also female hair. Sexual relationship, 87.8% half a year had anal sex with men, 63.2% in the last time in the use of condoms in the anal intercourse, 16.8% months with women in sexual behavior, only 27.3% in the last time with women in the use of condoms, 4.6% sex trade, the way to find sex with the Internet, accounting for 79.0%.323 urine specimens. The infection rate of Mg was 4.3% (95%CI=3.1%-5.4%), and the factors associated with Mg infection were not found. The results showed that the rate of Mg infection was low in the MSM population, suggesting that Mg infection was mainly transmitted by heterosexual behavioral pathways, and MSM was more significant as the source of Mg infection.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R759
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