建立PCR-CE-RFLP方法快速诊断女性生殖道的细菌感染

Study of rapid identification of pathogenic bacteria on feminine genital tract by PCR-CE-RFLP

目的建立PCR-CE-RFLP方法直接检测女性生殖道感染标本中的细菌,为临床提供快速、准确的诊断依据。方法利用细菌16S rRNA集保守性和变异性于一体的特点,合成1对通用引物,其上游5′端用5-′FAM标记。将8株已知菌和38例临床诊断为生殖道炎症的标本及其培养物进行PCR扩增,扩增产物经限制性内切酶HaeⅢ消化,经毛细管电泳(CE)通过限制性片断长度多态性分析(RFLP)来鉴定不同病原菌。结果(1)已知菌株PCR扩增阳性率为75%(6/8)、生殖道感染标本的PCR扩增阳性率为79%(30/38),毛细管电泳阳性率两者均为100%。(2)已知菌株PCR扩增产物的5′端HaeⅢ完全酶切片段的毛细管电泳结果,与电子克隆产物的酶切片段基本一致。(3)用传统培养检测法在生殖道能检出的常见致病菌,实验用PCR-CE-RFLP法同样也能检出。(4)实验还发现数种传统培养法未检出的细菌。结论PCR-CE-RFLP法比传统的细菌培养法具有快速、准确、灵敏的优点,比传统方法缩短20 h,可用于临床标本的直接鉴定。

Objective In order to provide with rapid and accurate evidence for clinical diagnosis through directly testing bacteria on feminine genital tract specimin by using PCR-CE-RFLP. And to develop genie CMOS cttip for testing urinary infective bacteria for clinical diagnosis. Methods A pair of universal primer of bacteria with 5＇-FAM on upstream primer were synthesized according to high conservative sequence of bacteria 16S rDNA. 8 known strains and 38 uncultured samples from inflammatory diseases of genital tract directly PCR,at the same time the latter were cultured then PCR. After that,the PCR productions were all digested by Hae Ⅲ ,and their digested productions were analyzed and characterized by Restriction-Fragment-Length Polymorphism（RFLP） after CE. Results （ 1 ） The positive rate reaction of reserved strains and uncultured samples on PCR reactin were respectively 75 % （6/8） and 79% （30/38）. The positive rate of CE were individually 100% （6/6,30/30）. （2）The PCR-CE-RFLP results from complete digestion of reserved strains were accorded with that of electronic-PCR. （3）Escherlchia coli, Stphylococcus epidermididis, Klebsierlla peumoniae and Acinetobacter baumannii could be detected from pathogenic bacteria on genital tract by PCR-CE-RFLP as well as routine method. （4）Several undetected bacteria by routine method could be detected by PCR-CE-RFLP. Condusion PCR-CE-RFLP was a more rapid,accurate and sensitive method than that of routine method, which was shorter 20 hours than the traditional mehod and could be applied on uncultured clinical samples.