【作者】 张楠；

【导师】 刘运德；

【作者基本信息】 天津医科大学 ， 临床检验诊断学， 2013， 硕士

【摘要】 目的探讨解脲脲原体(Ureaplasma urealyticum, Uu)、微小脲原体(Ureaplasma parvum, Up)感染与男性非淋菌性尿道炎(nongonococcal urethritis, NGU)的相关性,通过系统性回顾分析,揭示在男性非淋菌性尿道炎中Uu, Up感染对疾病的作用,为鉴别检测Uu和Up提供依据。通过将传统的PCR检测方法与微孔板核酸杂交以及酶显色技术相结合,建立起用于Uu, Up检测和鉴别的PCR-ELISA方法,并对该方法进行初步的临床应用性评价。方法采用Meta分析的方法,检索2000-2011年公开发表的有关Uu,Up感染与男性非淋菌性尿道炎的文献,并应用Review Manager5.0软件进行综合定量评价,计算其优势比(OR)等。应用生物素标记的U4,U5引物进行PCR扩增,利用Primer在线软件反向查出Uu,Up扩增出的核酸序列并根据NCBI在线Blast软件设计出Uu,Up特异性捕获探针,将捕获探针包被到DNA-BINDTM微孔板上并与PCR产物进行杂交,引入辣根过氧化物酶标记的链霉亲和素检测系统进行显色,从而初步构建出PCR-ELISA检测方法。通过对生殖支原体G37、β-溶血性链球菌、大肠杆菌、绿脓杆菌、金黄色葡萄球菌等参考菌和Uu代表菌株血清型8,Up代表菌株血清型3进行PCR-ELISA方法检测以验证该方法的特异性。同时随机选取了天津医科大学第二医院计划生育科不孕症检查的38例女性阴道拭子样本用于初步评估本方法的临床有效性。结果本次Meta分析共纳入中英文文献11篇包括病例组研究对象1824例和对照组研究对象1584例。脲原体(Uu+Up)总体感染率与NGU的关系合并OR值为1.23,95%CI(0.91,1.68),显著性检验P=0.18。其中Uu感染与NGU的关系OR值为1.65,95%CI(1.10,2.47,),显著性检验P=0.02,而Up感染与NGU的关系OR值为0.57,95%CI(0.45,0.72),显著性检验P<0.00001。本研究自行设计了用于检测鉴别Uu和Up的捕获探针,序列如下,Uu捕获探针：5’AGC AAT TAA CTT CGC TGAAGG C3’; Up捕获探针：5’GGC AAT TAA TTT CGC TAG TGG T3’。同时利用U4,U5引物对,Uu, Up捕获探针和DNA-BINDTM微孔板等初步构建出PCR-ELISA法用于检测和鉴别Uu,Up。在特异性实验中,本法可较好的区分检测Uu和Up,并且不与其他参考菌发生非特异性扩增或杂交反应。随后,应用38例随机选取的女性阴道拭子样本,初步评估了所建PCR-ELISA方法的临床有效性,其中检出阳性样本26例,包括Up感染21例,Uu感染3例,Uu, Up共感染2例,阴性结果12例,阳性检出率为68.42%。本方法与传统的培养法相比,应用Kappa检验进行一致性验证,Kappa值为0.687。以培养法为检测标准则PCR-ELISA法敏感度为88.89%(24/27),特异度为81.82%(9/11)。结论Uu主要分布在NGU组中,与Up相比具有更高的致病力,是男性非淋菌性尿道炎的致病因子之一；而Up则有相对较低的毒性,多以寄居的形式存在。因此,进行Uu和Up区分检测对于流行病学调查和致病机制的研究将具有重要意义。本研究将PCR扩增、微孔板核酸杂交和酶显色技术相结合初步构建出PCR-ELISA检测方法用于检测和鉴别Uu, Up。与传统培养法相比,PCR-ELISA法能有效的进行脲原体的检测并可同时鉴别出Uu和Up。本方法操作简单,无需特殊的仪器设备并且所需试剂均为商品化产品。本研究所建立的PCR-ELISA法通过一对特异性捕获探针将Uu和Up检测和鉴别出来,与以往的PCR-ELISA法相比无需引入地高辛及其抗体,不仅减少了操作步骤,而且成本经济效应较好,适合推广到各类基层医学微生物实验室。本法是一种有效的Uu, Up检出和鉴别工具,可作为目前临床Uu, Up检测的重要补充方法。更多还原

【Abstract】 ObjectiveInvestigate the correlation between Ureaplasma urealyticum, Ureaplasma parvum infection and male nongonococcal urethritis. By the retrospective study, explore the Uu, Up different virulence in the male nongonococcal urethritis, which will provide the important reason for developing a method, which can detect and differentiate the Ureaplasmas at the same time. Accoeding to what is said above, we attempt to combines the PCR, nucleic acid hybridization and enzyme color technology and establish a technology named PCR-ELISA to detect and differentiate the Ureaplasma urealyticum, Ureaplasma parvum.MethodsThe studies on the relationship of Ureaplasmas infection and male nongonococcal urethritis were collected by searching from Pubmed database,CBM database,CNKI database and so on(2000-2011), and analyzed by meta-analysis. The odds ratio was calculated by Review Manager5.0software. In the second section, we use the biotin-labelled primer pair (U4, U5) amplified the Ureaplasma urealyticum and Ureaplasma parvum by polymerase chain reaction (PCR). Search for the PCR product’s nucleic acid sequence by the Primer online program and designed the special capture probes to Ureaplasma urealyticum and Ureaplasma parvum. Bind the capture probes to the DNA-BINDTM microplate. The biotin-labelled PCR denatured products were hybridized with the capture probes. Then the biotin reacted with the HRP-Streptavidin system and a colorimetric assay was performed. Last, integrated the procedures and established the PCR-ELISA method. Meanwhile, the specificity testing was determined by using the reference bacterias such as Mycoplasma genitalium G37, Beta hemolytic streptococcus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus and Up standerd strain serotype3, Uu standard strain serotype8. In addition, the38clinical swabs were randomly selected from the Second Hospital of Tianjin Medical University to assess the method’s clinical feasibility.ResultsEleven studies accorded with research criteria including1824cases and1584 controls were analyzed. The combined OR value of Ureaplasmas infection to male NGU is1.23,95%CI(0.91,1.68), the test for overall effect P=0.18.However, the OR value of Uu infection to male NGU is1.65,95%CI(1.10,2.47), the test for overall effect P=0.02. But, contrary to Uu, the OR value of Up infection to NGU is0.57,95%CI(0.45,0.72), the test for overall effect P<0.00001. In the second section, the capture probes to differentiate the Ureaplasma urealyticum and Ureaplasma parvum were designed as followed:Uu capture probe:5’AGC AAT TAA CTT CGC TGA AGG C3’; Up capture probe:5’GGC AAT TAA TTT CGC TAG TGG T3’. Soon afterwards, the PCR-ELISA method was preliminarily established by applying the U4, U5primer pair, the capture probe and the DNA-BINDTM microplate. The method was specific so that only the Uu and Up can be detected and discriminated in the specificity testing. Meanwhile in the clinical feasibility testing, the PCR-ELISA method succeeded to detect and differentiate the38clinical swabs. Among the38clinical swabs, the positive specimens were26, comprising of the Up infections21, Uu infections3, Up, Uu co-infections2and negative specimens12. The positive rate was68.42%. Compared to conventional culture, the Kappa value was0.687. There was a certain extent consistency between the two methods. Meanwhile, using culture method as a standard, the PCR-ELISA method’sensitivity is88.89%(24/27), the specificity81.82%(9/11).ConclusionsUu mainly exists in the NGU group. Contrast to Up, Uu has higher virulence and is the one of male NGU virulent agents. However, Up has the lower virulence and is the parasitic microorganism in the male urethra. So, it is important to detect separately Ureaplasma urealyticum and Ureaplasma parvum. It will have far-reaching influence for epidemiological survey and mechanism study. The PCR-ELISA assay is a technology, which combines the PCR, nucleic acid hybridization and enzyme color technology. The PCR-ELISA can not only effectively dectet the Ureaplasmas like the culture method, but also differentiate the Uu and Up. The procedure is simple, the reagent is easily obtained from the commercial products and no special machine is needed. In this study, only a pair of capture probe is used to discriminate the pathogenic microorganism. Compared to previous PCR-ELISA, the digoxin and anti- digoxin antibody are not needed, so this not only reduce the procedure but also lower the cost. The PCR-ELISA method is suit to be applied to the secondary hospital and simple microorganism laboratory. The PCR-ELISA method is complemental to culture method and it will provide a new approach for the detecting and differentiating of Ureaplasma urealyticum and Ureaplasma parvum.更多还原