双色荧光定量聚合酶链反应诊断生殖器溃疡性疾病病因的实验研究及临床应用评价  

Experimental study and clinical application of double color fluorescent quantitative polymerase chain reaction for the identification of the etiology of genital ulcer disease among patients attending STD clinics in Guangzhou

作　　者：朱慧兰[1] 胡斌[2] 李润祥[1] 林路洋[1] 熊斯颖[1] 赖维[3] 

机构地区：[1]广州市皮肤病防治所,广东广州510095 [2]中山大学达安基因诊断中心,广东广州510080 [3]中山大学附属三院皮肤科,广东广州510630

出　　处：《中国艾滋病性病》2008年第2期151-153,共3页Chinese Journal of Aids & STD

基　　金：广东省科技计划项目基金(项目编号:2005B34201018)

摘　　要：目的建立检测梅毒螺旋体(TP)、单纯疱疹病毒(HSV)和杜克雷嗜血杆菌(HD)的双色荧光定量聚合酶链反应(DC-FQ-PCR)方法,并评价其在诊断生殖器溃疡性疾病(GUD)病因中的临床价值。方法根据TP、HSV和HD的基因库序列设计引物,对3株参考株进行扩增,证实其特异性;再设计探针做三种参考株的双色荧光定量PCR;以10倍稀释不同浓度模板扩增测定其敏感度。同时检测320份生殖器溃疡处分泌物标本,每份标本同时进行暗视野显微镜(D-F)、梅毒血清学试验(STS)、HD培养和酶免疫法(EIA)检查HSV抗原。结果用FQ-M-PCR检测TP、HSV和HD 3株参考株均出现特异性扩增片段,扩增片段大小分别为227bp、233bp和238bp;其敏感度为10拷贝/μl。将DC-FQ-PCR用于检测320例生殖器溃疡分泌物标本,与D-F、TPHA、EIA法比较,有较好的一致性(Kappa值分别为0.758、0.797、0.703)。结论DC-FQ-PCR是一种高灵敏度、高特异性和高精确性的检测生殖器溃疡标本中TP、HSV、HD的方法,可用于临床GUD的病因诊断。Objective To develop a method of double color fluorescent quantitative polymerase chain reaction detection （DC-FQ-PCR）of Treponema pallidum, Herpes Simplex Virus Types1/2, and Haemophilus ducreyi from genital ulcers among patients attending STD clinics in Guangzhou, and to evaluate the clinical application of this method for the identification of the etiology of genital ulcer diseases（GUD）. Methods Primers were designed with reference to the DNA sequence of T.pallidum, HSV1/2 and H.ducreyi.The specificity of the primers was confirmed in three reference strains of T. pallidum, HSV1/2 and H. ducreyi. The probe was designed to test the three reference strains in DC-FQ-PCR assay. The template was serially diluted at 1 ： 10 to test the sensitivity of DC-M-PCR. The developed method was used to test 320 samples from genital ulcers. Each sample was examined by dark-field examination（D-F） ,serology tests for syphilis（STS）, enzyme immunoassay（EIA）for HSV antigen and culture for H. ducreyi. Results Specific 227bp, 233bp and 238bp products were produced from the three reference strains of T. pallidum, HSV1/2 and H. ducreyi by DC-FQ-PCR. The sensitivi- ty of DC-FQ-PCR was 10 copies per microliter. DC-M-PCR assay for T. pallidum, HSV1/2 and H ducreyi showed good agreement as compared with D-F detection for T. pallidum STS. H. ducreyi culture and E1A for HSV1/2 antigen（Kappa scores are 0.758,0. 797,0.703, respectively）. Conclusions The DC-FQ-PCR is a convenient, accurate and reliable assay for the detection of T. pallidum. HSV1/2 and H. ducreyi from genital ulcers, and can be used as a tool for the identification of the etiology of GUD.

关 键 词：双色荧光定量聚合酶链反应 生殖器溃疡性疾病 梅毒螺旋体 单纯疱疹病毒 杜克雷嗜血杆菌 

分 类 号：R795[医药卫生—临床医学]