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
向琳 1陈雨珊 1晏嘉 2徐令清 2田国宝 3钟兰兰 3鲁洋 2李林海1
作者信息
- 1. 广州中医药大学研究生院,广东 广州 510405;广州医科大学附属清远医院(清远市人民医院)检验科,广东清远 511518
- 2. 广州医科大学附属清远医院(清远市人民医院)检验科,广东清远 511518
- 3. 中山大学中山医学院,广东 广州 528406
- 折叠
摘要
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NTsys 2.10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1 µl,模板DNA 0.75 μl,2×PCR Taq Mix 9 μl,ddH2O 8.25 μl;最佳扩增参数为Tm=38 ℃,退火40 s,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.
Abstract
OBJECTIVE To explore the application of random amplified polymorphic DNA(RAPD)typing effect in Klebsiella pneumoniae and acid-producing Klebsiella oxytoca.METHODS Totally 22 strains of acid-producing Klebsiella oxytoca 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 re-producibility experiments between amplification instruments and within amplification batches were performed af-terwards.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,0.75 μl of template DNA,9 μl of 2 × PCR Taq mix,and 8.25 μl of ddH2O;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 com-bination was RAPD1+M13,which distinguished Klebsiella pneumoniae from acid-producing Klebsiella oxytoca 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 oxytoca by RAPD,which was easy to operate,had good reproducibility and stability,and was of great signifi-cance for the molecular epidemiologic classification of pathogenic microorganisms.
关键词
随机扩增多态DNA技术/肺炎克雷伯菌/产酸克雷伯菌/细菌分型技术Key words
Random amplified polymorphic DNA technique/Klebsiella pneumoniae/Acid-producing Klebsiella oxytoca/Bacterial typing techniques引用本文复制引用
基金项目
广东省医学科学技术研究基金资助项目(A2021490)
清远市科技计划基金资助项目(2022808114560471)
清远市人民医院医学科研基金资助项目(202301-201)
清远市人民医院医学科研基金资助项目(318)
清远市人民医院医学科研基金资助项目(2023-研-008)
出版年
2024
NETL
NSTL
万方数据