产气荚膜梭菌四环素耐药基因双重荧光定量PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.10.034

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4630-4637.doi: 10.11843/j.issn.0366-6964.2024.10.034

产气荚膜梭菌四环素耐药基因双重荧光定量PCR检测方法的建立

欧兰欣¹^,²(), 叶碧锦¹^,², 孙铭飞², 戚南山², 李娟², 吕敏娜², 林栩慧², 蔡海明², 胡俊菁², 宋勇乐², 陈祥杰², 朱易斌², 尹理君², 张健騑², 廖申权²^,*(), 张浩吉¹^,*()

1. 佛山科学技术学院生命科学与工程学院, 佛山 528225
2. 广东省农业科学院动物卫生研究所广东省畜禽疫病防治研究重点实验室农业农村部禽流感等家禽重大疾病防控重点实验室, 广州 510640

收稿日期:2023-10-26 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 廖申权,张浩吉 E-mail:760903362@qq.com;lsq6969@163.com;zhanghaoji@aliyun.com
作者简介:欧兰欣(2000-), 女, 湖南宜章人, 硕士生, 主要从事动物传染病病因和致病机理研究, E-mail: 760903362@qq.com
基金资助:
广东省基础与应用基础研究基金项目(2021B1515120006);广东省科技计划项目(2021B1212050021);猪禽种业全国重点实验室开放课题(2023QZ-NK05);猪禽种业全国重点实验室开放课题(2022GZ07);广州市科技计划项目(2023B04J0137);广州市科技计划项目(2023A04J0789);科技创新战略专项资金(高水平农科院建设)(202110TD);科技创新战略专项资金(高水平农科院建设)(202122TD);科技创新战略专项资金(高水平农科院建设)(R2020PY-JC001);科技创新战略专项资金(高水平农科院建设)(R2019YJ-YB3010);科技创新战略专项资金(高水平农科院建设)(R2020PY-JG013);科技创新战略专项资金(高水平农科院建设)(R2020QD-048);科技创新战略专项资金(高水平农科院建设)(R2021PY-QY007);科技创新战略专项资金(高水平农科院建设)(R2023PY-JG018);广东省现代农业产业技术体系创新团队建设专项(2022KJ119);广东省农业科学院协同创新中心项目(XTXM202202)

Establishment of a Duplex Real-time PCR Method for Tetracycline Resistance Gene Detection in Clostridium perfringens

Lanxin OU¹^,²(), Bijin YE¹^,², Mingfei SUN², Nanshan QI², Juan LI², Minna LÜ², Xuhui LIN², Haiming CAI², Junjing HU², Yongle SONG², Xiangjie CHEN², Yibin ZHU², Lijun YIN², Jianfei ZHANG², Shenquan LIAO²^,*(), Haoji ZHANG¹^,*()

1. College of Life Science and Engineering, Foshan University, Foshan 528225, China
2. Key Laboratory of Avian Influenza and Other Major Poultry Diseases Prevention and Control, Ministry of Agriculture and Rural Affairs, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China

Received:2023-10-26 Online:2024-10-23 Published:2024-11-04
Contact: Shenquan LIAO, Haoji ZHANG E-mail:760903362@qq.com;lsq6969@163.com;zhanghaoji@aliyun.com

摘要/Abstract

摘要：

旨在开发一种快速、灵敏的针对产气荚膜梭菌四环素类耐药基因tetA(P)和tetB(P)的双重荧光定量PCR检测方法，为快速检测产气荚膜梭菌四环素类抗生素耐药性提供新策略。本研究针对tetA(P)和tetB(P)的保守序列设计引物和探针，构建重组质粒，对退火温度和体系进行优化，同时采用药敏纸片琼脂扩散法测定菌株对四环素类药物的敏感性加以验证。结果发现，优化后的反应程序为95 ℃ 30 s；95 ℃ 5 s，56 ℃ 30 s，共39个循环。使用该反应程序仅能扩增重组质粒，对照菌株无扩增曲线；组内变异系数小于1.3%，组间变异系数小于1.8%，重组质粒最低检测限度为10² copies·μL^-1。使用本方法对33份产气荚膜梭菌临床分离株样品进行耐药基因检测，同时进行药敏试验，发现本研究建立的检测方法对四环素耐药基因的检出率均为75.8%(25/33)，且与药敏试验结果一致。综上，本研究建立了一种特异、敏感、重复性好的产气荚膜梭菌四环素类耐药基因tetA(P)和tetB(P)的双重荧光定量PCR检测方法。

关键词: 产气荚膜梭菌, 四环素, tetA (P), tetB (P), 双重荧光定量PCR

Abstract:

The purpose of this study was to establish a rapid and sensitive duplex real-time PCR method for tetA(P) and tetB(P) tetracycline resistance genes detection in Clostridium perfringens, aiming at providing a new strategy for the expedited identification of tetracycline antibiotic resistance within C. perfringens. Primers and probes were designed to target the conserved sequences of tetA(P) and tetB(P), construct recombinant plasmid, and optimize the annealing temperature and system. At the same time, validation of the method was substantiated through disc diffusion test for the determination of strain of tetracycline drugs sensitivity. The results showed that, the optimized reaction program was 95 ℃ 30 s; 95 ℃ 5 s; 56 ℃ 30 s, 39 cycles. And this method exclusively amplified the recombinant plasmid with no amplification curve observed for control strains. Intra-assay and inter-assay coefficients of variation were observed to be less than 1.3% and 1.8%, respectively. The plasmid standard had a minimum detection threshold of 10² copies·μL^-1. Using this method, resistance gene detection was performed on 33 clinical isolates of C. perfringens, complemented by antimicrobial susceptibility testing. The detection method established by the current study yielded a detection rate of 75.8% (25/33) for tetracycline resistance genes, aligning significantly with the antimicrobial susceptibility testing results. In conclusion, this study successfully established a specific, sensitive, and highly reproducible duplex real-time PCR method for the detection of tetA(P) and tetB(P) tetracycline resistance genes in C. perfringens.

Key words: Clostridium perfringens, tetracycline, tetA(P), tetB(P), duplex real-time PCR

中图分类号:

S852.616.3

欧兰欣, 叶碧锦, 孙铭飞, 戚南山, 李娟, 吕敏娜, 林栩慧, 蔡海明, 胡俊菁, 宋勇乐, 陈祥杰, 朱易斌, 尹理君, 张健騑, 廖申权, 张浩吉. 产气荚膜梭菌四环素耐药基因双重荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2024, 55(10): 4630-4637.

Lanxin OU, Bijin YE, Mingfei SUN, Nanshan QI, Juan LI, Minna LÜ, Xuhui LIN, Haiming CAI, Junjing HU, Yongle SONG, Xiangjie CHEN, Yibin ZHU, Lijun YIN, Jianfei ZHANG, Shenquan LIAO, Haoji ZHANG. Establishment of a Duplex Real-time PCR Method for Tetracycline Resistance Gene Detection in Clostridium perfringens[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4630-4637.

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参考文献 21

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基因 Gene	引物和探针 Primer and probe	序列 Sequence	产物大小/bp Product size
tetA(P)	tetA-F	5′-AGGTGGACCAATTATAGGAATCA-3′	145
	tetA-R	5′-ACCTCCAACTCTATCATCCACC-3′
	tetA-P	HEX-AGCTACAAATATTTCAGTAAG-BHQ1
tetB(P)	tetB-F	5′-AGCAAAGCGGTATGGGATTT-3′	160
	tetB-R	5′-TCACAAACATTCCTCTACCTTCTGT-3′
	tetB-P	FAM-AACTATGAGGGCAACCTTT-BHQ1

质粒 Plasimd	浓度/(copies·μL^-1) Concentration	组内变异试验 Intra-group variation test		组间变异试验 Inter-group variation test
质粒 Plasimd	浓度/(copies·μL^-1) Concentration	平均数 ${\rm{\bar X}}$±SD	变异系数/% CV	平均数 ${\rm{\bar X}}$±SD	变异系数/% CV
tetA	10⁷	19.4±0.26	1.30	19.5±0.36	1.8
	10⁶	22.5±0.26	1.10	22.6±0.20	0.8
	10⁵	22.7±0.29	1.10	25.7±0.39	1.5
tetB	10⁷	20.4±0.15	0.70	20.5±0.32	1.5
	10⁶	23.7±0.18	0.70	23.6±0.30	1.2
	10⁵	26.4±0.09	0.30	26.4±0.18	0.6

菌株毒素型 Toxintype	菌株数 Isolates	耐药菌株数 Resistance strains			双重qPCR检测结果 Duplex real-time PCR test results		普通qPCR检测结果 Normal real-time PCR test results
菌株毒素型 Toxintype	菌株数 Isolates	四环素 Tetracycline	多西环素 Doxycycline	米诺环素 Minocycline	tetA(P)	tetB(P)	tetA(P)	tetB(P)
A	31	26/31	24/31	23/31	24/31	24/31	24/31	24/31
G	2	1/2	1/2	1/2	1/2	1/2	1/2	1/2

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产气荚膜梭菌四环素耐药基因双重荧光定量PCR检测方法的建立

Establishment of a Duplex Real-time PCR Method for Tetracycline Resistance Gene Detection in Clostridium perfringens

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