多重PCR-反向线点杂交技术在检测女性念珠菌和阴道毛滴虫感染中的应用  

Multiplex PCR-based Reverse Line Hybridization Assay for Simultaneous Detection of Trichomonas Vaginalis and the Common Candida

作　　者：向华国 熊礼宽[2] 樊超 黄玉佳 赵良玉 

机构地区：[1]深圳市福永医院,广东深圳518103 [2]深圳市慢性病防治院性病防治中心,广东深圳518020

出　　处：《中国皮肤性病学杂志》2009年第8期529-531,共3页The Chinese Journal of Dermatovenereology

基　　金：深圳市科技计划项目(200803205);深圳市宝安区科技计划项目(200803265)

摘　　要：目的建立和评价多重PCR-反向线点杂交技术(mPCR-RLB)快速同时检测阴道毛滴虫和念珠菌感染的方法。方法分别选择阴道毛滴虫特异性DNA重复序列设计一对特异性引物,以念珠菌属28srRNA至5.8s rRNA间隔序列Ⅱ(ITS2)为靶基因设计一对检测念珠菌的通用引物,构建二重PCR同时扩增阴道毛滴虫、白色念珠菌、热带念珠菌、克柔念珠菌、光滑念球菌、近平滑念珠菌、都柏林念株菌DNA,然后与通过氨基标记固定在尼龙膜上的各特异性寡核苷核探针杂交,并对女性阴道试子标本进行检测。结果多重PCR可同时扩增阴道毛滴虫、白色念珠菌、热带念珠菌、克柔念珠菌、光滑念球菌、近平滑念珠菌、都柏林念株菌临床或标准菌株DNA,上述念珠菌标准菌株可扩增出302～441bp DNA片段,阴道毛滴虫临床株可扩增出262bp DNA片段。6种念珠菌和阴道毛滴虫的特异性寡核苷酸探针可分别与其相应的PCR产物杂交。通过对150例女性阴道试子进行检测,mPCR-RLB检测念珠菌的阳性率为47.33%,阴道毛滴虫的阳性率为6.67%,二者同时阳性率为1.33%。而培养法检测念珠菌阳性率为36.00%,阴道毛滴虫的阳性率为2.00%,二者同时阳性率为0.67%。明显高于培养法(P<0.05)。结论多重PCR-RLB可快速同时检测念珠菌和阴道毛滴虫,为女性阴道炎的临床诊断提供了一种可靠的方法。Objective To study and evaluate the multiplex PCR-based reverse line blot hybridization macroarray （mPCR- RLB） in the detection of Trichomonas vaginalis and common Candida. Methods The DNAs from Trichomonas vaginalis and 6 species of Candida （ C. alblcans, C. troptcalis, C. krusei, C. glabrata, C. parapsilosis and C. dubliniensis ） were amplified with two sets of primers directed to repeat DNA fragment of Trichomonas vaginalis and the ITS2 region of the gene from 28s rRNA to 5.8s rRNA of Candida respectively. The reverse primers were labeled with biotin. The products were identified by hybridization with its specific oligonucleotide probes labeled with amidogen in a nylon membrane. The method was applied in the detection of the swab specimens. Results All of Trichomonas vaginalis and 6 species of Candida strains were amplified by mPCR-RLB, the length of Candida and Trichomonas vaginalis PCR products was 302 -441bp and 262bp. These probes proved specific （they hybridized with only their target molecules）. The method was used to test 150 vaginal swabs. The positive rate of mPCR-RLB for Candida and Trichomonas vaginalis was higher than that in culture （P 〈 0.05 ）. Conclusion The mPCR-RLB is a simple, fast method for simultaneous detection of Candida and Trichomonas vaginalis infections, and it plays a very important role in the diagnosis of STI.

关 键 词：聚合酶链反应 核酸杂交 阴道毛滴虫 念珠菌 寡核苷核探针 

分 类 号：R394[医药卫生—医学遗传学] R537.7[医药卫生—基础医学]