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

欧兰欣 ¹叶碧锦 ¹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³

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作者信息

1. 佛山科学技术学院生命科学与工程学院,佛山 528225;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
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
3. 佛山科学技术学院生命科学与工程学院,佛山 528225
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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.

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 perfrin-gens,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 se-quences 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 ob-served 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 thresh-old of 102 copies·μL-1.Using this method,resistance gene detection was performed on 33 clini-cal isolates of C.perfringens,complemented by antimicrobial susceptibility testing.The detec-tion method established by the current study yielded a detection rate of 75.8％(25/33)for tetra-cycline resistance genes,aligning significantly with the antimicrobial susceptibility testing re-sults.In conclusion,this study successfully established a specific,sensitive,and highly repro-ducible duplex real-time PCR method for the detection of tetA(P)and tetB(P)tetracycline re-sistance genes in C.perfringens.

关键词

产气荚膜梭菌/四环素/tetA(P)/tetB(P)/双重荧光定量PCR

Key words

Clostridium perfringens/tetracycline/tetA(P)/tetB(P)/duplex real-time PCR

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基金项目

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)

出版年

2024

畜牧兽医学报

中国畜牧兽医学会

畜牧兽医学报

CSTPCD北大核心

影响因子：0.729

ISSN：0366-6964

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