TaqMan-小沟结合物荧光探针对青海省青南地区104株鼠疫菌链霉素耐药基因rpsL突变位点的筛查  

Screening of rpsL mutations in streptomycin resistance gene of 104 strains of Yersinia pestis strains in south area of Qinghai Province by TaqMan-MGB fluorescent probe

作　　者：柏吉祥[1] 辛有全[1] 李胜[1] 靳娟[1] 张琪[1] 杨晓艳[1] 金泳[1] 彭文轩 代瑞霞[1] 何建[1] BAI Ji-xiang;XIN You-quan;LI Sheng;JIN Juan;ZHANG Qi;YANG Xiao-yan;JIN Yong;PENG Wen-xuan;DAI Rui-xia;HE Jian(Specialized Laboratory of Plague Bacteria,Qinghai Institute for Endemic Disease Control and Prevention,Xining,Qinghai 810021,China)

机构地区：[1]青海省地方病预防控制所鼠疫菌专业实验室,青海西宁810021

出　　处：《中国热带医学》2023年第6期662-666,共5页China Tropical Medicine

基　　金：国家自然科学基金项目(No.82260401);青海省科技厅基础研究计划项目(No.2019-ZJ-7074)。

摘　　要：目的了解青海省青南地区rpsL基因点突变引起鼠疫菌耐链霉素的现状,为今后青南地区突发人间鼠疫的精准临床用药及预防耐药发生提供理论依据。方法筛选1957—2009年青海省青南地区取自鼠疫患者、媒介昆虫体内及中间宿主的104株代表性鼠疫菌,用赫氏平皿进行分离培养,采用十二烷基磺酸钠裂解和苯酚-氯仿法提取代表性鼠疫菌的DNA,针对我国链霉素耐药基因rpsL基因设计引物及TaqMan-小沟结合物(minor groove binder,MGB)探针Probe1[FAM]和Probe2[VIC],利用TaqMan-MGB荧光探针的实时荧光聚合酶链反应(polymerase chain reaction,PCR)技术,对青南地区104株鼠疫菌链霉素耐药位点rpsL基因的突变情况进行检测。结果青南地区104株被试菌株FAM检测结果均为阳性,对应检测rpsL(128:A),相对荧光单位(relative fluorescence unit,RFU)峰值>1000,阴性<200。所有被试菌株VIC检测结果均为阴性,对应检测rpsL(128:G),RFU峰值<200,阳性>1000。即青南地区104株鼠疫菌中未发现耐链霉素rpsL基因点突变菌株。结论了解到青南地区鼠疫菌耐链霉素菌株分布情况,为青南地区预防耐药发生以及鼠疫菌临床治疗提供了基础数据。Objective To investigate the current status of streptomycin resistance of Yersinia pestis caused by point mutations of rpsL gene in Qinghai,so as to provide theoretical basis for precise clinical medication and prevention of drug resistance of human plague outbreak in South area of Qinghai Province in the future.Methods A total of 104 representative strains of Yersinia pestis collected from plague patients,vector insects and intermediate hosts in South area of Qinghai Province from 1957 to 2009 were screened,isolated and cultured by Hiss agar plates.The DNA of representative Yersinia pestis was extracted by sodium dodecyl sulfate lysis and phenol-chloroform method.The primers forward primer and reverse primer and TaqMan-MGB probes probe1[FAM]and probe2[VIC]were designed for the rpsL gene of streptomycin resistance gene in China.Real-time PCR with TaqMan-MGB fluorescent probe was used to detect the mutations of rpsL gene in streptomycin resistance locus of 104 strains of Yersinia pestis in South area of Qinghai Province.Results The FAM test results of 104 strains in South area of Qinghai Province were positive,corresponding to the detection of rpsL(128:A),RFU peak>1000,negative<200.VIC test results of all tested strains were negative,corresponding to the detection of rpsL(128:G),RFU peak<200,positive>1000.That is,no strains with rpsL gene mutation related to streptomycin resistance were found in the 104 strains of Yersinia pestis in Qingnan Province.Conclusion This study provides basic data on the distribution of streptomycin resistance of Yersinia pestis in South area of Qinghai Province,and lays a foundation for preventing the occurrence of drug resistance and clinical treatment of Yersinia pestis in South area of Qinghai Province.

关 键 词：鼠疫菌 TaqMan-小沟结合物荧光探针 链霉素 RPSL基因 筛查 

分 类 号：R378.6[医药卫生—病原生物学]