一种羊种布鲁氏菌复方新诺明耐药株荧光定量PCR检测方法的建立  

Establishment of a Quantitative Real-time PCR Detection Method for Trimethoprim-Sulfamethoxazole-resistance Strains of Brucella melitensis

作　　者：杨晓雯 宁文晴 周师众 袁雅琴 侯雪新[2] 丁家波[1] YANG Xiaowen;NING Wenqing;ZHOU Shizhong;YUAN Yaqin;HOU Xuexin;DING Jiabo(Key Laboratory of Animal Biosafe Risk Prevention and Control(North)of Ministry of Agriculture and Rural Affairs,Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China;National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;College of Veterinary Medicine,Shandong Agricultural University,Tai’an 271001,China;Key Laboratory of Jiangsu Preventive Veterinary Medicine,Key Laboratory of Avian Preventive Medicine,Ministry of Education,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所农业农村部动物生物安全风险预警及防控重点实验室(北方),北京100193 [2]中国疾病预防控制中心传染病预防控制所传染病溯源预警与智能决策全国重点实验室,北京102206 [3]山东农业大学动物医学院,泰安271001 [4]扬州大学兽医学院禽类预防医学教育部重点实验室,江苏省动物预防医学重点实验室,扬州225009

出　　处：《畜牧兽医学报》2025年第3期1465-1472,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：北京市自然科学基金项目(7222120);中央级公益性科研院所基本科研业务费专项(2023-YWF-ZYSQ-09);中国农业科学院科技创新工程重大科研任务(CAAS-ZDRW202410);中国农业科学院科技创新工程科学中心重点任务(CAAS-CSLPDCP-202403);中国农业科学院科技创新工程(ASTIP)(ASTIP-IAS-15)中国农业科学院科技创新工程重大科研任务(CAAS-ZDRW202410);中国农业科学院科技创新工程科学中心重点任务(CAAS-CSLPDCP-202403);中国农业科学院科技创新工程(ASTIP)(ASTIP-IAS-15)。

摘　　要：近年来,世界范围内已分离获得布鲁氏菌复方新诺明耐药株,快速检测样品中是否含有耐药株有助于指导临床用药。目前国内外尚无布鲁氏菌复方新诺明耐药株的快速检测方法。本研究旨在建立一种羊种布鲁氏菌复方新诺明耐受株的快速检测方法。基于前期的研究筛选最小抑菌浓度较高的我国羊种布鲁氏菌分离株,利用基因组测序分析全基因组SNPs并筛选SNPs位点,利用MGB探针建立荧光定量检测羊种布鲁氏菌复方新诺明耐药株。研究发现,羊种布鲁氏菌2型标准株可作为耐药株分析的参考株,且64个SNPs和InDels为耐药株特有;12个突变位点可用于建立耐药株快速检测方法;利用染色体1中2014308位点的A/G多态性建立荧光定量检测方法特异性良好,最低能检测2×10^(1)CFU菌量,可用于临床样品是否含有复方新诺明耐药株的检测。本研究建立了一种羊种布鲁氏菌复方新诺明耐药株的荧光定量检测方法,为我国布病耐药株的防控提供科学依据,同时也为临床用药提供参考。In recent years,strains of Brucella spp.resistant to trimethoprim-sulfamethoxazole have been isolated worldwide.Rapid detection of these resistant strains in samples can guide clinical medication.Currently,there are no rapid detection methods for Brucella trimethoprim-sulfamethoxazole-resistance strains.The aim of this study is to establish a rapid detection method for trimethoprim-sulfamethoxazole-resistance strains of B.melitensis.Based on previous studies,the isolates of B.melitensis with higher minimum inhibitory concentrations were selected.Whole genome sequencing was used to analyze SNPs and InDels,and variation sites were selected for developing a quantitative real-time PCR detection method using MGB modified probes.The results showed that B.melitensis bv.2 strain 63/9 could be the reference genome to analysis resistance strains,and 64 unique SNPs and InDels have been passed.Among them,twelve mutation sites were identified that could be used to establish a rapid detection method for resistant strains.A quantitative real-time PCR detection method developed using the A/G polymorphism at site 2014308 of chromosome 1 showed good specificity and could detect as low as 2×101 CFU,making it suitable for detecting trimethoprim-sulfamethoxazole-resistance strains in clinical samples.In conclusion,this study established a quantitative real-time PCR detection method for trimethoprim-sulfamethoxazole-resistance B.melitensis strains,providing a scientific basis for the prevention and control of drug resistant strains in China,and can also serve as a reference for clinical medication.

关 键 词：羊种布鲁氏菌 复方新诺明 全基因组SNPs MGB探针 

分 类 号：S852.614[农业科学—基础兽医学]