白假丝酵母菌最优化RNA提取策略的探索

Strategy of Optimizing RNA Extraction from Candida Albicans

目的探索白假丝酵母菌总RNA简单、快捷、高效的提取方法。方法选取昆明医科大学第一附属医院妇产科门诊2011年7月—2013年3月有临床症状且阴道分泌物镜检阳性的外阴阴道假丝酵母菌病(VVC)患者的白假丝酵母菌40株,选用不同时间与温度组合下的4种溶壁酶(Lyticase)破壁方案(A方案:37℃、12 h,B方案:25℃、12 h,C方案:37℃、24 h,D方案:25℃、24 h)进行RNA提取,将所得RNA进行荧光定量PCR(q PCR)反应验证RNA的浓度及纯度,并进行管家基因18S rRNA的检测。结果 D方案提取获得的RNA浓度高于A、B、C方案(P<0.05);B方案提取获得的RNA浓度高于A、C方案(P<0.05);而A、C两方案提取获得的RNA浓度比较,差异无统计学意义(P>0.05)。B、D方案获得的RNA纯度高于A、C方案(P<0.05);但A、C方案获得的RNA纯度比较,差异无统计学意义(P>0.05);B方案提取获得的RNA纯度高于D方案(P<0.05)。A、B、C、D方案提取的RNA扩增产物Ct转化值比较,差异有统计学意义(P<0.05);其中B、D方案q PCR扩增产物Ct转化值高于A、C方案(P<0.05);但B、D方案及A、C方案之间的q PCR扩增产物Ct转化值比较,差异无统计学意义(P>0.05)。管家基因18S rRNA的引物扩增出的产物片段大小为150 bp。结论以Lyticase破壁提取白假丝酵母菌具有简单易行及RNA产量高、纯度好的优点,值得临床推广应用。

Objective To explore a simple,rapid and effective solution of extracting RNA from Candida albicans （C. albicans）. Methods From july 2011 to March 2013,in the First Affiliated Hospital of Kunming Medical University,40 isolates simulated from vaginal secretion were identified as C. albicans. This study chose 4 Lyticase disrupting - wall programs at different time and temperatures（program A：37 ℃ ,12 h;program B：25 ℃ ,12 h;program C：37 ℃ ,24 h;program D：25 ℃ ,24 h） to extract RNA. The concentrations and purity of the extracted RNA were verified by real - time fluorescent quantitative PCR（qPCR）reaction and housekeeping gene 18S rRNA detection performed. Results The RNA concentration of program D was higher than those of programs A,B,C,the difference was significant（ P 〈 0. 05）,that of program B was higher than those of programs A,C（P 〈 0. 05）,but there was no difference between programs A,C（P 〉 0. 05）. The RNA purity programs B,D was higher than those of programs A,C（P 〈 0. 05）,but there was no difference between programs A,C （P 〉 0. 05）. RNA purity of program B was higher than that of program D（P 〈 0. 05）. There was difference in RNA amplified product Ct conversion value in programs A,B,C,D（P 〈 0. 05）,there into the qPCR amplified product Ct conversion value of programs B,D was higher than that of programs A,C（P 〈 0. 05）,but there was no difference between programs B,D and A,C（P 〉 0. 05）. The product fragment size amplified by housekeeping gene 18S rRNA primers was 150 bp. Conclusion Lyticase wall - disruption extacting C. albicans is simple,easy to operate,with high RNA products and good purity,which is worthy of promotion.