作　　者： (何其励）; (王欣）; (黄娟）; (陈军剑）; (郭主声）; (张丽华）; (朱学海）; (杨维青）;

机构地区： 广东医科大学医学检验研究所临床微生物学教研室,广东东莞523808 四川省疾病预防控制中心毒理检验所,四川成都610041

出　　处： 《微生物学免疫学进展》 2017年第4期27-32,共6页

摘　　要： 目的建立快速鉴定B群链球菌(group B Streptococcus,GBS)的同时检测其对克林霉素、红霉素耐药基因的多重PCR方法,并初步应用。方法建立多重PCR方法,同时扩增以下4个基因位点:GBS编码CAMP因子的基因cfb、大环类脂类耐药基因erm B和mef A/E、克林霉素耐药基因lin B。对该方法的引物质量浓度、Taq酶量和退火温度进行优化,确定多重PCR最适反应体系及最低检测限;将该方法用于91株GBS临床菌株的鉴定及其耐药性的检测,并评价其与常规PCR方法结果的一致性。结果多重PCR的最适反应体系,总量25μL:多重PCR mix212.5μL、多重PCR mix1Taq酶0.23μL、lin B-F和lin B-R引物0.4μmol/L、mef A/E-F和mef A/E-R引物0.2μmol/L、erm B-F和erm B-R引物0.12μmol/L、cfb-F和cfb-R引物0.08μmol/L、DNA 10~100 ng、不足量补入无菌去离子水。扩增程序为:94℃预变性60 s;94℃变性30 s、53℃退火90 s、72℃延伸30 s,共30个循环;72℃延伸10 min。最低检测限为20 pg/μL。多重PCR方法与常规PCR方法检测91株GBS临床菌株,cfb基因结果一致率为100%,erm B、mef A/E和lin B基因的Kappa值分别为0.938、0.956和0.935。结论建立的以GBS cfb、erm B、mef A/E和lin B基因为检测位点的多重PCR方法,可在鉴定GBS的同时检测其对克林霉素、红霉素的耐药性。 Objective To set up a new multiplex PCR method which was used in identification of group B Streptococcus（GBS） and related resistance to clindamycin and erythromycin,and the method was preliminary applied. Methods Multiplex PCR method was designed to amplify 4 important genes loci： cfb gene encoding the CAMP factor presented in every GBS; macrolide resistance genes erm B and mef A/E; clindamycin resistance gene lin B. Primer concentration,annealing temperature and the optimal amount of enzyme were optimized. The optimal reaction system and the minimum detection limit of the method were determined,which was applied in identification of 91 GBS clinical isolated strains and detection of drug resistance for the strains,and evaluation of consistency of the tested result with that of conventional single PCR method. Results The total volume of the optimal multiplex PCR reaction was 25 μL,consisted of 12.5 μL of Mixtiplex pcr mix 2,0.23 μL of Mixtiplex pcr mix 1 Taq enzyme,0. 4 μmol/L of lin B primer pair,0. 2 μmol/L of mef A/E primer pair,0. 12μmol/L of erm B primer pair,0.08μmol/L of cfb primer pair,1μL of template DNA,and sterile deionized water was added to the specified volume. The reaction parameters were as following,an initial denaturation at 94 ℃ for 60 s,followed by 30 cycles of94 ℃ for 30 s,53 ℃ for 90 s,and 72 ℃ for 30 s,and a final10 minutes of 72 ℃ for extension. The minimum detection limit was 20 pg/μL. In comparison with the result of conventional single PCR,the consistency of the multiplex PCR for detection of cfb gene was 100%. Kappa values of the two methods in detection of erm B,mef A/E and lin B genes were 0.938,0.956 and 0.935,respectively. Conclusion The multiplex PCR method,which was used in identification of loci cfb,erm B,mef A/E and lin B genes,could be performed in detection of GBS resistance to erythromycin and clindamycin too.

关 键 词： 多重聚合酶链式反应 群链球菌 耐药基因 快速鉴定