应用TaqMan-MGB探针RT-qPCR技术对青海省海南州鼠疫菌耐链霉素rpsL基因突变位点的检测

doi:10.11853/j.issn.1003.8280.2023.03.009

摘要/Abstract

摘要： 目的研究青海省海南藏族自治州（海南州）鼠疫疫源地鼠疫耶尔森菌（鼠疫菌）对链霉素的敏感性,以便在突发鼠疫疫情中能精准地指导鼠疫临床用药。方法根据鼠疫菌基因序列设计特异性引物,以S19960127菌株核糖体蛋白S12编码基因（rpsL基因）突变位点和野生型鼠疫菌株rpsL基因位点分别设计探针,利用TaqMan-MGB探针技术对海南州1954－2009年分离的87株鼠疫菌进行耐链霉素rpsL基因突变位点检测。结果 87株被试菌株的野生型A碱基的羧基荧光素（FAM）探针检测结果均为阳性,每个反应管内荧光信号达到设定的阈值时所经历的循环数（Ct）值在18.74～21.93,相对荧光单位（RFU）峰值>2 000；S19960127的Ct值为25.42,RFU峰值<200；阴性对照RFU峰值<200。所有被试菌株突变型G碱基的亚磷酰胺（VIC）探针检测结果均为阴性,Ct值在20.04～24.79,RFU峰值<200；S19960127的Ct值为17.56,RFU峰值>1 000；阴性对照RFU峰值<200。实验结果显示海南州鼠疫疫源地87株鼠疫菌基因序列第128位的碱基未发生A→G突变,该疫源地未检出耐链霉素鼠疫菌株。结论海南州鼠疫疫源地87株鼠疫菌对链霉素均敏感。鉴于细菌耐药性基因的产生与传播特征,应持续开展监测并建立鼠疫菌耐药性监测网络,以便能及时发现并控制耐药菌的传播。

关键词: 鼠疫耶尔森菌, 耐链霉素, rpsL基因, 实时荧光定量PCR, TaqMan-MGB探针

Abstract: Objective To study the sensitivity of Yersinia pestis to streptomycin in natural plague foci of Hainan Tibetan Autonomous Prefecture (Hainan prefecture), Qinghai province, China, and to provide guidance on accurate medication use in case of plague emergencies.Methods Specific primers were designed based on the gene sequence of Y. pestis. Probes were designed for the mutant locus of the gene encoding the ribosomal S12 protein (rpsL gene) of the S19960127 strain and the rpsL gene site of the wild-type Y. pestis strain. TaqMan-MGB probes were used to detect streptomycin-resistant rpsL gene mutations in 87 strains of Y. pestis that were isolated in Hainan prefecture from 1954 to 2009.Results All the 87 strains were positive for wild-type A base detection with carboxyfluorescein probes; the number of cycles required for the fluorescent signal to cross the threshold in each reaction tube (Ct) ranged from 18.74 to 21.93, and the relative fluorescence unit (RFU) peak was >2 000; for S19960127, the Ct value was 25.42, and the RFU peak was <200; the RFU peak of the negative control was <200. All the tested strains were negative for mutant G base detection with VIC probes, the Ct value ranged from 20.04 to 24.79, and the RFU peak was <200; for S19960127, the Ct value was 17.56, and the RFU peak was >1 000; the RFU peak of the negative control was <200. The results showed that A-to-G base mutation was not detected at 128 bp of the gene sequence of the 87 Y. pestis strains in the plague foci of Hainan prefecture, indicating that no streptomycin-resistant Y. pestis strains were found in the plague foci.Conclusions All the 87 strains of Y. pestis from the plague foci of Hainan prefecture were sensitive to streptomycin. In view of the generation and transmission characteristics of bacterial resistance genes, continuous streptomycin resistance surveillance and establishment of a surveillance network should be carried out in order to timely detect and control streptomycin-resistant Y. pestis strains from being transmitted.

Key words: Yersinia pestis, Streptomycin resistance, rpsL gene, Real-time quantitative PCR, TaqMan-MGB probe

中图分类号:

R378.6⁺1

李胜, 靳娟, 何建, 张琪, 杨晓艳, 辛有全, 赵海红, 张晓璐, 柏吉祥, 代瑞霞. 应用TaqMan-MGB探针RT-qPCR技术对青海省海南州鼠疫菌耐链霉素rpsL基因突变位点的检测[J]. 中国媒介生物学及控制杂志, 2023, 34(3): 331-335.

LI Sheng, JIN Juan, HE Jian, ZHANG Qi, YANG Xiao-yan, XIN You-quan, ZHAO Hai-hong, ZHANG Xiao-lu, BAI Ji-xiang, DAI Rui-xia. Detection of streptomycin-resistant rpsL gene mutations in Yersinia pestis in Hainan prefecture, Qinghai province, China using quantitative real-time PCR with TaqMan-MGB probes[J]. Chinese Journal of Vector Biology and Control, 2023, 34(3): 331-335.

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