变性梯度凝胶电泳检测阴道菌群实验方法的建立及优化

Establishment and optimization of a procedure for denaturing gradient gel electrophoresis to detect vaginal microbiota

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月存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V2V3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。

Objectives To establish and optimize a procedure for culture-independent polymerase chain reaction denaturing gradient gel electrophoresis （PCR-DGGE） in order to analyze vaginal microbiota. Methods This study used PCR- DGGE to explore the vaginal microbiota of 16 women who were seen by Peking University Cancer Hospital from May to June 2011. Swab samples were obtained from the vaginal fornix and the middle third of the vagina in 10 of the 16 women. Only a vaginal fornix sample was obtained from the remaining 6 women. Bacterial DNA was extracted from the samples and nested PCR was used to amplify the DNA fragment of the V2 V3 hypervariable region of the bacterial 16S rRNA gene. DGGE was performed on a polyacrylamide gel formed with 8% stock solution containing a 30--60% or 40 70% denaturing gradient of urea and formamide that increased in the direction of electrophoresis. Results Gel Compare II software was used to perform pairwise comparison of the DGGE profiles of vaginal microbiota from the vaginal fornix and the middle third of the vagina, and this comparison indicated a high degree of similarity and marginal discrepancies. In the DGGE profiles, there was an average of 2.5 bands from the vaginal fornix and middle third of the vagina, and differences in the number of bands was not statistically significant （P =0. 072）. DGGE profiles of vaginal microbiota that were obtained on polyacrylamide gels with an 8% stock solution containing a 30%- 60% denaturing gradient of urea and for mamide were clear and the bands were readily identifiable. However, some marker bands fell outside the gel. DGGE pro files obtained on polyacrylamide gel with an 8% stock solution containing a 400%- 70% denaturing gradient of urea and formamide were also clear and the bands in each lane were also readily identifiable. No bands fell outside the gel. Conclusion PCR-DGGE is a powerful molecular tool to analyze vaginal microbiota.