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目的建立并优化应用变性梯度凝胶电泳(DGGE)检测阴道菌群的技术路线。方法 2011年5~6月在北京大学肿瘤医院妇科门诊就诊的16名成年女性,其中6名女性取阴道穹隆分泌物,10名女性分别取阴道中段和阴道穹窿部分泌物,提取阴道菌群总DNA,采用巢式聚合酶链式反应扩增细菌16SrRNA基因的V2-V3可变区,DNA扩增产物分别在30%~60%和40%~70%浓度梯度变性凝胶下进行DGGE。结果 DGGE图像分析显示阴道中段和阴道穹窿分泌物阴道菌群构成相同,平均条带数为2.5,差异无统计学意义(P>0.05)。变性凝胶浓度梯度为30%~60%时,阴道菌群的DGGE图像中各条带较分散,易于条带识别和分析,但有部分条带电泳出凝胶范围;变性凝胶浓度梯度为40%~70%时,阴道菌群的DGGE条带密集但均较清晰。结论巢式聚合酶链式反应与DGGE相结合具有高效、快速、精确的特点,可用于阴道菌群检测。
Objective To establish and optimize the technique of denaturing gradient gel electrophoresis (DGGE) for the detection of vaginal flora. Methods From June to June 2011, 16 adult women were referred to Gynecology Clinic of Peking University Cancer Hospital. Six women took vaginal vault secretions and 10 women took the vaginal vault for the vaginal fornix. The total vaginal flora DNA, the V2-V3 variable region of bacterial 16S rRNA gene was amplified by nested polymerase chain reaction. The DNA amplification products were subjected to DGGE under denaturing gradient gels of 30% -60% and 40% -70% respectively. Results The results of DGGE showed that the vaginal flora in the middle vagina and vaginal vault secretions had the same composition with an average number of bands of 2.5, with no significant difference (P> 0.05). When the concentration gradient of denaturing gel was 30% -60%, the bands in DGGE images of vaginal flora were more dispersed, which was easier to identify and analyze the bands. However, some bands electrophoresed out the gel range. The denaturing gel concentration gradient was 40% ~ 70%, the vaginal flora of DGGE bands are dense but are more clear. Conclusion The combination of nested polymerase chain reaction and DGGE has the characteristics of high efficiency, rapidity and precision and can be used for the detection of vaginal flora.