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

doi:10.11843/j.issn.0366-6964.2025.03.044

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (3): 1465-1472.doi: 10.11843/j.issn.0366-6964.2025.03.044

• 研究简报 • 上一篇

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

杨晓雯¹^,²(), 宁文晴¹^,³(), 周师众¹, 袁雅琴¹^,⁴, 侯雪新²^,*(), 丁家波¹^,*()

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

收稿日期:2024-04-16 出版日期:2025-03-23 发布日期:2025-04-02
通讯作者: 侯雪新,丁家波 E-mail:yangxiaowen01@caas.cn;m17662631372@163.com;houxuexin@icdc.cn;dingjiabo@126.com
作者简介:杨晓雯(1989-)，女，山东潍坊人，研究员，博士，主要从事人兽共患病防控、耐药及致病机制研究，E-mail: yangxiaowen01@caas.cn
宁文晴(2000-)，女，山东济南人，硕士生，主要从事人兽共患病防控、耐药及致病机制研究，E-mail: m17662631372@163.com
第一联系人：
杨晓雯和宁文晴为同等贡献作者
基金资助:
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)

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

YANG Xiaowen¹^,²(), NING Wenqing¹^,³(), ZHOU Shizhong¹, YUAN Yaqin¹^,⁴, HOU Xuexin²^,*(), DING Jiabo¹^,*()

1. 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
2. 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
3. College of Veterinary Medicine, Shandong Agricultural University, Tai'an 271001, China
4. 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

Received:2024-04-16 Online:2025-03-23 Published:2025-04-02
Contact: HOU Xuexin, DING Jiabo E-mail:yangxiaowen01@caas.cn;m17662631372@163.com;houxuexin@icdc.cn;dingjiabo@126.com

摘要/Abstract

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2 014 308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2×10¹ 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。

关键词: 羊种布鲁氏菌, 复方新诺明, 全基因组SNPs, MGB探针

Abstract:

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 2 014 308 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.

Key words: Brucella melitensis, trimethoprim-sulfamethoxazole, whole genome SNPs, MGB modified probe

中图分类号:

S852.614

杨晓雯, 宁文晴, 周师众, 袁雅琴, 侯雪新, 丁家波. 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[J]. 畜牧兽医学报, 2025, 56(3): 1465-1472.

YANG Xiaowen, NING Wenqing, ZHOU Shizhong, YUAN Yaqin, HOU Xuexin, DING Jiabo. Establishment of a Quantitative Real-time PCR Detection Method for Trimethoprim-Sulfamethoxazole-resistance Strains of Brucella melitensis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1465-1472.

图/表 5

表 1

图 1

图 2

表 2

图 3

参考文献 23

1	NELSON-JONES A . Brucellosis[J]. Postgrad Med J, 1952, 28 (324): 529- 534. doi: 10.1136/pgmj.28.324.529
2	BUKHARI E E . Pediatric brucellosis. An update review for the new millennium[J]. Saudi Med J, 2018, 39 (4): 336- 341. doi: 10.15537/smj.2018.4.21896
3	PAPPAS G , PAPADIMITRIOU P , AKRITIDIS N , et al. The new global map of human brucellosis[J]. Lancet Infect Dis, 2006, 6 (2): 91- 99. doi: 10.1016/S1473-3099(06)70382-6
4	LIU Z G , GAO L P , WANG M , et al. Long ignored but making a comeback: a worldwide epidemiological evolution of human brucellosis[J]. Emerg Microbes Infect, 2024, 13 (1): 2290839. doi: 10.1080/22221751.2023.2290839
5	WANG H Y , LIU H Y , ZHANG Q R , et al. Natural history of and dynamic changes in clinical manifestation, serology, and treatment of brucellosis, China[J]. Emerg Infect Dis, 2022, 28 (7): 1460- 1465.
6	《中华传染病杂志》编辑委员会. 布鲁菌病诊疗专家共识[J]. 中华传染病杂志, 2017, 35 (12): 705- 710. doi: 10.3760/cma.j.issn.1000-6680.2017.12.001
	Editorial Board of Chinese Journal of Infectious Diseases . Expert consensus on the diagnosis and treatment of brucellosis[J]. Chinese Journal of Infectious Diseases, 2017, 35 (12): 705- 710. doi: 10.3760/cma.j.issn.1000-6680.2017.12.001
7	DESHMUKH A , HAGEN F , SHARABASI O A , et al. In vitro antimicrobial susceptibility testing of human Brucella melitensis isolates from Qatar between 2014- 2015[J]. BMC Microbiol, 2015, 15, 121.
8	BARBOSA PAULETTI R , REINATO STYNEN A P , PINTO DA SILVA MOL J , et al. Reduced susceptibility to rifampicin and resistance to multiple antimicrobial agents among Brucella abortus isolates from cattle in Brazil[J]. PLoS One, 2015, 10 (7): e0132532. doi: 10.1371/journal.pone.0132532
9	TORKAMAN ASADI F , HASHEMI S H , ALIKHANI M Y , et al. Clinical and diagnostic aspects of brucellosis and antimicrobial susceptibility of Brucella isolates in Hamedan, Iran[J]. Jpn J Infect Dis, 2017, 70 (3): 235- 238. doi: 10.7883/yoken.JJID.2016.133
10	DADAR M , ALAMIAN S , BRANGSCH H , et al. Determination of virulence-associated genes and antimicrobial resistance profiles in Brucella isolates recovered from humans and animals in Iran using NGS technology[J]. Pathogens, 2023, 12 (1): 82. doi: 10.3390/pathogens12010082
11	杨晓雯, 姜霞, 安翠红, 等. 羊种布鲁氏菌分离株体外抗生素敏感性研究[J]. 疾病监测, 2021, 36 (12): 1286- 1290.
	YANG X W , JIANG X , AN C H , et al. Sensitivity of Brucella melitensis isolates to antibiotics in vitro[J]. Disease Surveillance, 2021, 36 (12): 1286- 1290.
12	杨晓雯, 韩秀瑞, 朴东日, 等. 一株羊种变异布鲁氏菌分离株遗传特征分析[J]. 中国人兽共患病学报, 2020, 36 (11): 894- 899.
	YANG X W , HAN X R , PIAO D R , et al. Genetic characteristics of the Brucella melitensis strain with a rough phenotype[J]. Chinese Journal of Zoonoses, 2020, 36 (11): 894- 899.
13	PRJIBELSKI A , ANTIPOV D , MELESHKO D , et al. Using SPAdes de novo assembler[J]. Curr Protoc Bioinformatics, 2020, 70 (1): e102.
14	DARLING A C E , MAU B , BLATTNER F R , et al. Mauve: multiple alignment of conserved genomic sequence with rearrangements[J]. Genome Res, 2004, 14 (7): 1394- 1403.
15	BOUNAADJA L , ALBERT D , CHÉNAIS B , et al. Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes[J]. Vet Microbiol, 2009, 137 (1-2): 156- 164.
16	SKÖLD O . Sulfonamide resistance: mechanisms and trends[J]. Drug Resist Updat, 2000, 3 (3): 155- 160.
17	田国忠, 崔步云, 赵鸿雁, 等. 一起羊种布鲁氏菌病暴发的流行病学和分子特征研究[J]. 疾病监测, 2017, 32 (3): 211- 215.
	TIAN G Z , CUI B Y , ZHAO H Y , et al. Studies on epidemiology and molecular characteristics of Brucella melitensis during an outbreak of brucellosis[J]. Disease Surveillance, 2017, 32 (3): 211- 215.
18	梁英风, 吴金英, 刘岩, 等. 62株临床分离布鲁氏菌对四种常用抗生素药物敏感性实验[J]. 现代检验医学杂志, 2020, 35 (4): 116-117, 129.
	LIANG Y F , WU J Y , LIU Y , et al. Sensitivity test of 62 strains of Brucella isolated from clinical to four common antibiotics[J]. Journal of Modern Laboratory Medicine, 2020, 35 (4): 116-117, 129.
19	MA H R , XU H J , WANG X , et al. Molecular characterization and antimicrobial susceptibility of human Brucella in Northeast China[J]. Front Microbiol, 2023, 14, 1137932.
20	YUAN H T , WANG C L , LIU L N , et al. Epidemiologically characteristics of human brucellosis and antimicrobial susceptibility pattern of Brucella melitensis in Hinggan League of the Inner Mongolia Autonomous Region, China[J]. Infect Dis Poverty, 2020, 9 (1): 79.
21	刘志国, 王妙, 李晓燕, 等. 内蒙古乌兰察布地区布鲁氏菌体外药物敏感性及耐药机制分析[J]. 中国人兽共患病学报, 2019, 35 (5): 393- 398.
	LIU Z G , WANG M , LI X Y , et al. In vitro drugs susceptibility and resistance mechanism investigation of Brucella isolates from Ulanqab region of Inner Mongolia[J]. Chinese Journal of Zoonoses, 2019, 35 (5): 393- 398.
22	LIU Z G , DI D D , WANG M , et al. In vitro antimicrobial susceptibility testing of human Brucella melitensis isolates from Ulanqab of Inner Mongolia, China[J]. BMC Infect Dis, 2018, 18 (1): 43.
23	赵忠智, 崔步云, 姜海, 等. 人源布鲁菌的药敏试验研究[J]. 中华地方病学杂志, 2019, 38 (7): 536- 540.
	ZHAO Z Z , CUI B Y , JIANG H , et al. Drug sensitivity test of human derived Brucella[J]. Chinese Journal of Endemiology, 2019, 38 (7): 536- 540.

菌株 Strains	组装后长度/bp Assembly	N50长度/bp N50 length	contigs数量 Number	最大contigs长度/bp Max contigs	测序深度(×) Coverage (×)	可用reads比例/% Used reads ratio
RSXT015	3 288 879	298 831	26	609 534	234	97.60
RSXT019	3 288 710	296 830	26	609 502	230	97.01
RSXT089	3 288 684	296 861	26	609 517	229	95.69

序号 Number	序列(5′→3′) Sequence	功能 Function
seq1	5′-TCTTTGTGGGCGGCTATCC-3′	扩增布鲁氏菌检测序列 Amplification sequences of Brucella spp.
seq2	5′-CCGTTCGAGATGGCCAGTT-3′
seq3	5′Cy5-ACGGGCGCAATCT-3′BHQ-1	布鲁氏菌检测探针 Probe for Brucella spp. detection
seq4	5′- CAGGATTTTCCGATCGACCTT-3′	扩增耐药株检测序列 Amplification sequences of resistance strain
seq5	5′- AAGCTGTTCTAAGCGGCACATAG-3′
seq6	5′-VIC- CGGTGGCGACAGA-3′ MGB	敏感株检测探针 Probe for sensitive strain detection
seq7	5′-FAM- TGGCGGCAGATAT-3′ MGB	耐药株检测探针 Probe for resistance strain detection

[1]	马春玲, 任善会, 杨雪, 蔺玉刚, 李积雲, 王相伟, 殷相平, 孙跃峰, 万学瑞, 陈豪泰. 基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立[J]. 畜牧兽医学报, 2024, 55(4): 1800-1809.
[2]	王韬;唐辉;曾勇庆;李晓宁;闫超;董林松;于希江. 应用MGB荧光探针技术确定猪KIT基因拷贝重复数[J]. 畜牧兽医学报, 2011, 42(3): 437-441.

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 23

相关文章 2

编辑推荐

Metrics

本文评价