RAPD在肺炎克雷伯菌及产酸克雷伯菌分型中的应用

Application of RAPD in the typing of Klebsiella pneumoniae and acid-producing Klebsiella oxytoca

目的探索随机扩增多态DNA技术(RAPD)在肺炎克雷伯菌及产酸克雷伯菌分型中的应用。方法收集2022年12月-2023年10月清远市人民医院临床分离的产酸克雷伯菌22株、肺炎克雷伯菌74株,通过筛选模板、单引物、RAPD反应体系及扩增参数、双引物,构建克雷伯菌分型体系,后进行聚合酶链反应(PCR)扩增仪器间、扩增批次内(间)重复性实验。对96株已知克雷伯菌确定分型相似度标准,运用NTsys2.10软件构建遗传距离聚类图。结果煮沸提取基因组DNA满足实验需要,可在-20℃、60d条件下稳定保存;筛选出7条单引物用于双引物配对组合,最佳双引物反应体系为:引物各1μl,模板DNA0.75μl,2×PCR Taq Mix9μl,ddH_(2)O 8.25μl最佳扩增参数为Tm=38℃,退火40s,循环30次;重复性实验获得的RAPD图谱结果基本一致。分型效果最好的双引物组合为RAPD1+M13,该引物组合可在86%的相似度下区分肺炎克雷伯菌与产酸克雷伯菌的差异,将96株克雷伯菌分为40种亚型。结论上述双引物组合可初步用于RAPD对肺炎克雷伯菌及产酸克雷伯菌的种间分型,操作简便,具有较好重复性和稳定性,对病原微生物的分子流行病学分类具有重要意义。

OBJECTIVE To explore the application of random amplified polymorphic DNA(RAPD)typing effect in Klebsiella pneumoniae and acid-producing Klebsiella orytoca.METHODS Totally 22 strains of acid-producing Klebsiella orytoca and 74 strains of Klebsiella pneumoniae were collected from Dec.2022 to Oct.2023 from the clinical isolation of Qingyuan People's Hospital,and the typing system of Klebsiella was constructed by selecting templates,single primers,RAPD reaction system and amplification parameter,and double primers,and the reproducibility experiments between amplification instruments and within amplification batches were performed afterwards.The typing similarity criteria were determined for 96 known Klebsiella strains,and the genetic distance clustering diagram was constructed using NTsys 2.10 software.RESULTS The genomic DNA was extracted by boiling to meet the experimental needs,and could be stably stored at-20℃and 60 d.Seven single primers were selected for the double primer pair combination,and the optimal double-primer reaction system was as follows:1μl of each primer,0.75μl of template DNA,9μl of 2×PCR Taq mix,and 8.25μl of ddH_(2)O;and the optimal amplification parameters were Tm=38℃,annealing for 40 s,and cycling for 30 times.The results of the RAPD profiles obtained from the repeatability experiments were basically consistent.The best typing double primer combination was RAPD1+M13,which distinguished Klebsiella pneumoniae from acid-producing Klebsiella orytoca at 86%similarity and classified 96 Klebsiella strains into 40 subtypes.CONCLUSION The above double primer combination could be initially used for interspecies typing of Klebsiella pneumoniae and acid-producing Klebsiella orytoca by RAPD,which was easy to operate,had good reproducibility and stability,and was of great significance for the molecular epidemiologic classification of pathogenic microorganisms.