反相斑点杂交法对解脲脲原体分型的研究

Rapid detection and identification of Ureaplasma urealyticum species by PCR reversed dot blot

目的研究以聚合酶链反应为基础的快速检测与鉴定解脲脲原体基因型的方法。方法选择2003年11月至2005年11月在中山大学附属第二医院门诊就诊的有外阴阴道炎症状和体征的患者601例,设为病例组,同期无自觉症状的正常体检人群306例,设为对照组,分别取宫颈分泌物待检测。将解脲脲原体不同基因型的特异探针固定在硝酸纤维素膜上,临床标本按常规方法提取解脲脲原体DNA,采用生物素标记的解脲脲原体特异通用引物PCR扩增DNA,然后分别与解脲脲原体不同基因型特异探针杂交、显色。结果病例组解脲脲原体阳性421例占70.0%,对照组解脲脲原体阳性126例占41.2%。病例组中单型别感染的U.parvum占65.4%,其中1型、3型、6型和14型分别占28.8%、43.3%、26.0%和1.9%,U.urealyticum占18.4%;对照组中单型别感染的U.parvum占79.3%,其中1型、3型、6型和14型分别占63.2%、21.1%、15.7%和0.0%,U.urealyticum占13.8%。18例阳性标本随机DNA测序鉴定,均为相应的解脲脲原体基因型。结论U.parvum群,尤其是其中的1、3、6型别是正常人群携带的可能性较大,U.urealyticum则有可能和1型起协同作用或独自导致疾病。用反相斑点杂交进行解脲脲原体基因分型,方法简单、实用,适用于临床。

Objective To establish a rapid method for detection and identification of Ureaplasma urealyticum species by PCR-reversed dot blot from clinical specimens. Methods Six hundred and one lower genital tract infected women who were admitted to the second aff＇diated hospital of Sun Yat-Sen University from November 2003 to November 2005 were chosen as case group. Three hundred and six normal health examination women were set as control group. We took cervical secretions and extracted DNA for examining. The species-specific probes to identify Ureaplasma urealyticum were added NH2 tails and then inoculated on a nitrocellulose filter. The DNA from each isolated was amplified by PCR with the Uu-specific universal primers, and the PCR products labeled with bios-Ⅱ during amplification were hybridized with the species-specific probe. Results The positive rate of Uu in case group was 70.0%, while in control group was 41.2%. U. parvum was 65.4% in single genotype infection of case group. Of the total, genotype 1,3,6 and 14 was 28.8% ,43.3% ,26.0% and 1.9% ,respectively. U. urealyticum was 18.4%. U. parvum was 79.3% in control group. Genotype 1,3,6 and 14 was 63.2% ,21.1% ,15.7% ,0.0% ,respectively. U. urealyticum was 13.8%. Eighteen positive cases were chosen randomly for DNA sequencing and were proved to be correct. Conclusions People infected with single genotype （especially genotype 1, 3 and 6）of U. parvum may be normal carriers. U. urealyticum may be lead to infection by itself or cooperated with genotype 1. PCR-reversed dot blot is a rapid and simple method for detection and identification of Ureaplasma urealyticum species.