用PCR法快速半定量阴道分泌物中的乳酸杆菌

目的 通过对PCR引物、反应条件及样本处理条件的摸索,建立一种不经过分离培养和DNA提取纯化步骤,直接用乳酸杆菌特异性引物快速定性、半定量妇女阴道分泌物标本中乳酸杆菌的方法.方法 设计乳酸杆菌属特异性引物;对样本处理方法和PCR反应条件进行摸索;比较用PCR法定量阴道分泌物中乳酸杆菌的结果和稀释滴种法定量结果的相关性.结果 （1）用浓度为2%的Tritonx-100处理阴道标本后可在220 bp的位置得到最佳的特异性扩增区带;（2）将MgCl2在PCR反应体系中的终浓度调节至2.5 mmol/L时,可以产生最强的特异性反应扩增带;（3）用PCR法分析阴道分泌物所得菌数与培养所得菌数之间明显相关（P＜0.05）;用PCR法分析阴道分泌物所得灰度值与培养所得菌数之间明显相关（P＜0.05）.结论 将阴道分泌物标本经过简单的离心、洗涤处理后,直接应用PCR法,就可以快速地定性和半定量妇女阴道分泌物标本中的乳酸杆菌.

The study of detection and semi-quantification of the Lactobacilli in vaginal by PCR

Objective To use PCR we found a new method that could qualify and semi-quantify in vagina accurately and quickly without separating bacteria after vaginal sample centrifuge and washing.Methods We designed Lactobacilli genus-specific primers basing on 16S rRNA.We used PCR semi-quantifying Lactobacilli in vaginal samples without separating bacteria and DNA purifying after sample centrifuge and washing.We also compared the correlation between normal PCR Lactobacilli quantification and traditional diluting Lactobacilli quantification.Results (1) 2% Triton-X100 was the optimum concentration that can release DNA template;(2) When the concentration of MgCl_2 was to 2.5 mmol/L,the PCR product obtained was clearly;(3) There was no obvious difference between normal PCR quantification in vaginal sample and cultured bacteria(P>0.05);there was obvious correlation between normal PCR quantification in vaginal sample and cultured bacteria quantification(P<0.05).Conclusion Normal PCR can qualify and semi-quantify the Lactobacilli in woman vaginal sample quickly.