DNA芯片检测泌尿生殖道病原体及耐药的研究

目的 研制一种DNA芯片,结合多重PCR方法快速检测泌尿生殖道炎症3种病原体及其耐药类型。方法 根据泌尿生殖道病原体淋球菌、沙眼衣原体和解脲脲原体的基因保守序列设计引物和探针,分别检测3种病原体;根据淋球菌gyrA、parC和16srRNA基因序列突变位点设计引物和探针,分别检测淋球菌对喹诺酮类药物和大观霉素的耐药性;根据淋球菌和解脲脲原体的共同tetM基因序列设计引物和探针,检测对四环素的耐药性。制备芯片,对152份泌尿生殖道拭子提取DNA进行7重PCR扩增,并且Cy5荧光标记包含上述基因序列的目的DNA片段,与固定在芯片上的探针杂交,芯片信号分析系统Scanarray 4000在635nm处扫描并分析结果,并与临床检查结果比较。结果 DNA芯片敏感性是0.01fg质粒DNA。152份泌尿生殖道炎症拭子其病原体种类及其耐药类型全部可用DNA芯片检测出来,与目前临床检查结果有较好的一致性(K>0.8)。结论 研制的DNA芯片结合7重PCR方法快速检测泌尿生殖道淋球菌、沙眼衣原体和解脲脲原体3种病原体及其对喹诺酮类药物、大观霉素和四环素的耐药类型,具有快速、高特异性和高灵敏度,可以应用于临床检测。     

基因芯片技术检测生殖器溃疡性性病病原体

目的建立检测引起生殖器溃疡性性病病原体的基因芯片技术。方法设计并合成各病原体特异性探针，用点样仪将合成的特异探针点于玻片介质上，并经过点样后处理制备成基因芯片；荧光标记各病原体标准株及临床标本的特异靶基因，进行基因芯片杂交、扫描并分析结果。结果各病原体均可见特异的基因芯片荧光杂交信号：单一病原体出现一种特异荧光信号；混合病原体的不同组合出现相应组合的特异荧光信号。对40例生殖器溃疡临床标本进行芯片杂交检测，并与暗视野显微镜 聚合酶链反应(PCR)及HSV培养 PCR结果比较，具有较好的一致性(K值分别为0．882和0．947)。另外发现混合感染2例。结论本研究初步建立的基因芯片技术具有准确、特异及同时检测多重感染的特点。     

微孔板双探针杂交法快速鉴别都柏林念珠菌和白念珠菌

目的 建立微孔板双探针杂交法,快速鉴别都柏林念珠菌和白念珠菌,并应用于临床分离株的快速鉴定。方法 利用真菌保守区核糖体基因和可变区内转录间隔区基因为靶序列,应用生物素标记的通用引物进行念珠菌DNA的PCR扩增,并将该PCR产物分别与固定在微孔板上的都柏林念珠菌和白念珠菌特异性探针杂交,经酶联显色反应测其A值。结果 能特异地鉴别白念珠菌和都柏林念珠菌标准菌株。对108株常规培养方法从临床标本分离的白念珠菌检测,结果显示106株菌株仅白念珠菌探针检测阳性,另2株菌株仅都柏林念珠菌探针阳性。结论 微孔板双探针杂交法能快速、特异地鉴别都柏林念珠菌和白念珠菌。     

多重PCR结合反向线点杂交方法检测七种性病病原体

目的 建立多重PCR结合反向线点杂交技术同时检测淋球菌、 沙眼衣原体、生殖支原体、人型支原体、微小脲原体、解脲脲原体和毛滴虫的方法。方法 设计7对生物素标记的上述病原体特异性引物同时扩增病原体靶基因。扩增产物与预先标记在尼龙膜上的特异性探针杂交、显影，判读结果。多重PCR结合反向线点杂交方法检测211个尿道、宫颈分泌物临床标本（男107例，女104例），并与传统单一引物PCR检测结果比较，评价反向线点杂交的敏感性和特异性。结果 多重PCR结合反向线点杂交技术能敏感和特异地检测所有这7种病原体标准株，能敏感地检测病原体100拷贝的靶基因。在211个临床标本的检测中，2.8% （6 /211）的标本使用多重PCR结合反向线点杂交技术检测为阳性，而单一引物PCR检测为阴性，再使用巢式PCR检测这6个标本，结果为阳性。结论 多重PCR结合反向线点杂交技术能快速、敏感和特异地检测多种性病病原体。     

DNA芯片快速检测淋球菌gyrA基因突变

目的 研制一种新型DNA芯片,用于快速检测淋球菌gyrA基因突变。方法 根据淋球菌gyrA基因的序列信息设计探针并制作DNA芯片,PCR扩增并荧光标记包含gyrA基因突变的目的DNA片段,与芯片杂交,同时以测序法为对照。结果 50份泌尿生殖道拭子全部可用DNA芯片检测出来,芯片检测结果与药敏结果符合率为100%,与测序结果二者符合率为98%。结论 DNA芯片检测淋球菌gyrA基因突变具有快速、高特异性和高灵敏度,可以应用于临床耐药性检测。     

四种分枝杆菌快速检测方法的研究

目的 建立敏感、特异的快速检测4种分枝杆菌的方法。方法 用特异性引物对结核分枝杆菌、鸟分枝杆菌、胞内分枝杆菌和堪萨斯分枝杆菌菌悬液DNA进行PCR扩增,验证其敏感性和特异性。将上述4种分枝杆菌的特异性引物同时放入PCR扩增反应体系中,以此反应体系分别对该4种分枝杆菌菌悬液DNA、及其两两组合或特定的三重组合进行扩增,同时验证其敏感性。结果 4种分枝杆菌的特异性引物分别放入各自的PCR扩增反应体系中,可分别特异性地扩增出该4种分枝杆菌对应的DNA片段,敏感性达1×10¹～1×10²个菌细胞/mL。4种分枝杆菌的特异性引物同时放入同一PCR扩增反应体系中可分别特异性地扩增出对应的4种分枝杆菌单菌及其两两组合或三种分枝杆菌组合的DNA片段,敏感性达1×10²～1×10³个菌细胞/mL;4种分枝杆菌的特异性引物对其他分枝杆菌进行扩增,结果均为阴性。结论 多重PCR方法能敏感、特异地快速检测4种分枝杆菌。     

聚合酶链反应检测深部致病真菌的实验研究

目的 建立能用于临床实践的检测常见致病真菌的聚合酶链反应(PCR)方法.方法 设计了以热启动PCR为基础的实验方法,首先用真菌通用引物对标本进行单重PCR,若阳性,再用白念珠菌、烟曲霉和新生隐球菌的种特异性引物进行三重PCR来检测这3种常见致病真菌.结果 对9属55种78株常见深部真菌均扩增出260bp的DNA片段,而对细菌和人DNA均未扩增出目的 片段,具有高度特异性和敏感性,该方法操作简便且成本低.结论 以热启动PCR为基础的单重PCR和三重PCR方法可能成为临床上深部真菌感染理想的快速诊断工具.     

反向斑点杂交技术检测泌尿生殖道沙眼衣原体及基因分型

目的 建立反向斑点杂交技术,对泌尿生殖道沙眼衣原体感染进行检测和基因分型。方法 用Oligo软件设计寡核苷酸探针,采用巢式聚合酶链反应(PCR)扩增omp1基因的VS1-VS2序列,反向斑点杂交技术对沙眼衣原体进行检测和基因分型。结果 巢式PCR扩增omp1基因的VS1-VS2序列的敏感性与质粒PCR符合率为98.2%(56/57)。优选出11条型特异性(A、B+Ba、C、D、E、F、G、H、I、J和K)和3条群特异性寡核苷酸探针:B群(B、Ba、D和E型),C群(A、C、H、I、J和K型)和中间群(F和G型)。特异性经过基因库中的BLAST程序比较和试验优化,结果显示各探针均与标准株呈特异性杂交。56例VS1-VS2 PER阳性的临床标本杂交法检测均阳性,共检出59株沙眼衣原体,包括8个基因型,其中以E、F、D和H型为主,占77.9%,分别为25.4%,22.0%,16.9%和13.6%。发现3例混合感染占5.4%,分别为D/E、D/F和F/K。结论 反向斑点杂交技术简便且快速,可直接对临床标本沙眼衣原体进行检测和基因分型。     

皮肤分枝杆菌感染微孔板反向杂交检测法的研究

目的 探讨微孔板反向分子杂交技术与PCR-ELISA相结合的“拟芯片”法检测和鉴定几种常见的皮肤分枝杆菌感染的方法。方法 首先对5种分枝杆菌标准菌株(结核分枝杆菌、鸟分枝杆菌、胞内分枝杆菌、堪萨斯分枝杆菌和偶遇分枝杆菌)16s rRNA DNA进行PCR扩增获得其PCR产物:其次对"拟芯片"法反应条件进行优化;并对其敏感性、特异性进行检测;再用建立的方法对9例临床分离株进行检测和鉴定。结果 "拟芯片"法反应条件优化结果为:探针包被浓度为100 nmol/mL,探针包被时间为15 min,杂交的温度为55℃,杂交时间为45 min,辣根过氧化物酶标记的亲和素的稀释度为1:800;"拟芯片"法敏感性为1×10¹～1×10²个菌细胞/mL;特异性较好,各菌探针与其他分枝杆菌间无交叉。结论 "拟芯片"法是快速、敏感及特异的皮肤分枝杆菌感染诊断方法之一。     

马尔尼菲青霉临床分离株的rDNA ITS序列分析

目的 为设计马尔尼菲青霉种特异性引物,探讨马尔尼菲青霉病更加确切的诊断方法.方法 实验菌株为北京大学真菌和真菌病研究中心保存的4株马尔尼菲青霉,来源于国内不同地区.用真菌通用引物ITS1和ITS4 PCR扩增马尔尼菲青霉rDNAITS,扩增产物纯化后直接测序,测序结果在基因库核酸序列数据库进行同源序列搜索,并依据序列对比、分析.结果 4株临床分离的马尔尼菲青霉的rDNA ITS序列相同.与国外来源于美国、印度尼西亚、法国、澳大利亚的马尔尼菲青霉rDNA ITS序列基本一致.马尔尼菲青霉与荚膜组织胞浆菌、新生隐球菌、念珠菌的rDNAITS序列差异较大,青霉和曲霉属间rDNAITS的序列相似性较低,而青霉种间rDNAITS序列的差异不大.结论 不同来源的马尔尼菲青霉菌株间乃至不同青霉的种间的rDNA ITS序列均具有较高的同源性,提示该区可能不适于作为靶基因来设计马尔尼菲青霉的种特异性引物或探针.     

低密度基因芯片检测病毒性STDs病原体方法的建立

目的采用聚合酶链反应(PCR)结合反向杂交技术建立检测常见病毒性性病病原体的低密度基因芯片。方法将H IV-1,HSV-2,HPV6,HPV11的特异性寡核苷酸探针加尾并以一定阵列固定在尼龙膜上,经过处理后制成低密度芯片。在扩增过程中用B iotin-16-dUTP标记扩增产物,产物变性后分别与点有各种探针的尼龙膜反向杂交,杂交结果采用链霉亲和素-碱性磷酸酶(SP-AP)显色系统检测,并对检测体系的敏感性和特异性进行了测试。结果4种探针只与其对应的DNA杂交,而不和其他病毒杂交。敏感性试验可检出20 fg的病毒DNA。结论低密度基因芯片具有特异、敏感、快速等优点,采用低密度基因芯片一次杂交就可同时筛选鉴定多种性传播疾病(STD s)病毒的感染,值得临床推广应用。     

皮肤感染性肉芽肿常见病原菌微孔板反向杂交检测法的研究

目的 探讨微孔板反向分子杂交技术与PCR-ELISA相结合的“拟芯片”法检测和鉴定皮肤感染性肉芽肿常见病原菌的方法。 方法 选取、设计真菌和分枝杆菌两对通用引物,及10种皮肤感染性肉芽肿常见的病原菌：结核分枝杆菌、麻风分枝杆菌、海鱼分枝杆菌、偶遇分枝杆菌、脓肿分枝杆菌、申克孢子丝菌、卡氏枝孢霉、裴氏着色真菌、F. Monophora、白念珠菌的特异性探针;采用标准株验证通用引物、特异性探针并检测“拟芯片”法的敏感性、特异性;对19株临床分离株进行检测和鉴定。 结果 两对通用引物均可扩增出对应的菌种基因序列,PCR产物经测序分析与预期相符;“拟芯片”法检测阴性标本吸光度为0.041 ± 0.02,公式计算得临界值为0.101,在此基础上测得其敏感性为1 × 10 ~ 1 × 102个菌细胞/ml。各病原菌与相应的探针杂交,吸光度均 ＞ 0.101,为阳性;而与非对应的探针杂交,则吸光度 ＜ 0.101,为阴性,特异性高;对临床分离株的菌种鉴定准确率高。 结论 “拟芯片”法可应用于皮肤感染性肉芽肿常见病原菌的实验室快速检测和鉴定。     

Für die andrologische Diagnostik relevante sexuell übertragbare Infektionen

Affects on male fertility are associated with many sexually transmitted diseases. Genital tract infections play a major role in this context. The evidence for an impact on fertility differs for the pathogens; however early treatment may be very important. This requires fast and precise clinical diagnostics. Further, sexually transmitted infections may have major relevance in andrologic diagnostics because of the risk of transmission to the mother or fetus. Particularly for the increasingly relevant HIV and hepatitis infections, current guidelines are available for use in diagnostics and assisted reproduction techniques.     

Dermatovenerological infections

In dermatology, infections caused by bacteria, viruses, fungi, and parasites play an important role. A large proportion of pathogen-related infections of the skin and mucous membranes are transmitted sexually. All areas of infectious diseases and dermatovenerology are subject to highly exciting, dynamic change. This is driven by changes in the epidemiology of long-established diseases, changes in the resistance of pathogens to anti-infectives, recurrence of known pathogens, and the emergence of completely new pathogens. In this article, we address “resistance to anti-infectives”, “sexually transmitted infections”, and “emerging viral infections”, three core areas of dermatovenerology that will shape the field in the years to come.     

Herpes simplex virus seroprevalence and risk factors in 2 Canadian sexually transmitted disease clinics

OBJECTIVES: The objectives of this study were to determine the seroprevalence and risk factors for herpes simplex virus (HSV) types 1 and 2 in patients attending 2 Canadian sexually transmitted disease (STD) clinics. STUDY: Stored sera were tested for the presence of IgG class antibodies to HSV-1 and HSV-2 and results linked to that obtained from a risk behavior questionnaire. RESULTS: Overall prevalences for HSV-1 and -2 were 56% and 19%, respectively. HSV-1 and -2 seropositivity was associated with increasing age, female gender, nonwhite ethnicity, and a history of STD. HSV-2 seropositivity was also associated with a history of genital herpes, presence of genital sores, and coinfection with either human immunodeficiency virus (HIV) or hepatitis C (HCV). CONCLUSIONS: Herpes simplex infection is common in this high-risk Canadian population. Our finding that HCV seropositivity was a significant predictor for HSV-2 seropositivity emphasizes the overlap between pathogens that are primarily thought to be bloodborne pathogens and sexually transmitted infections and the need to target prevention in these areas concurrently.     

Prevalence of asymptomatic sexually transmitted infections among high-risk patients attending a free anonymous HIV-screening center

INTRODUCTION: The aim of this study was to assess the prevalence of sexually transmitted infections among patients attending an anonymous HIV Screening Center. PATIENTS AND METHODS: This prospective study was performed in the HIV Screening Center of University hospital in Reims (France) from May 1997 to December 1997. The inclusion criteria were the asymptomatic clinical presentation and the presence of risk factors for sexually transmitted infections referring to WHO criteria. The methods included clinical examination after application of acetic acid and urethral and endocervical swabs to identify:Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, Gardnerella vaginalis, Trichomonas vaginalis in specific culture. Treponema pallidum and HIV-1 infection were both detected by Enzym Linked Immuno Sorbent Assay (ELISA). RESULTS: One hundred and one patients (62 men and 39 women) were included in the study. Their mean age was 27 +/- 4 Years. Risk factors for sexually transmitted infections were: multiple sexual partners 81 p. 100; homo or bisexuality 16 p. 100; intravenous drug use 3 p. 100. The sexually transmitted infections were: HIV-1 infection 1 p. 100;Ureaplasma urealyticum 25 p. 100; genital warts 5 p. 100;Chlamydia trachomatis 3 p. 100; Gardnerella vaginalis 3 p. 100; Mycoplasma hominis 2 p. 100; Treponema pallidum 0 p. 100; Neisseria gonorrhoeae 0 p. 100; Trichomonas vaginalis 0 p. 100. The prevalence of sexually transmitted infections was significantly higher among women (p<0.05). DISCUSSION: Classical sexually transmitted infections and HIV infection were rarely detected in this study; but prevalence of other sexually transmitted infections (genital warts, Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum) was high. Ureaplasma urealyticum is considered as a possible pathogenic agent in pregnant women (preterm delivery, decrease of birth weight, chorioamniotitis). These results suggest that other than sexually transmitted infections in high risk patients attending a HIV Screening Center other sexually transmitted infections should also be systematically screened for.     

Prevalence of sexually transmitted disease infection in women alleging rape.

During a 2-year period ending in October, 1989, 110 women who claimed to have been raped were treated at the sexually transmitted disease clinic at Birmingham General Hospital. A total of 22 sexually transmitted infections were found in 14 (13%) women, of which Chlamydia trachomatis (in 8%) and Trichomonas vaginalis (in 6%) were the most frequently found pathogens. In none of these patients was it possible to determine whether infection resulted from rape or from voluntary intercourse with another person. The high prevalence of infection found in this study and in previous studies indicates that all women alleging rape should be investigated at a sexually transmitted disease clinic.