常见耐药基因多重PCR方法的建立及用于禽致病性大肠杆菌耐药性监测

doi:10.11843/j.issn.0366-6964.2020.12.029

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (12): 3193-3198.doi: 10.11843/j.issn.0366-6964.2020.12.029

常见耐药基因多重PCR方法的建立及用于禽致病性大肠杆菌耐药性监测

张耀东, 朱红, AFAYIBO Dossêh Jean Apôtre, 王瑶, 易正飞, 信素华, 陶程琳, 李涛, 祁晶晶, 田明星, 丁铲, 于圣青*, 王少辉*

中国农业科学院上海兽医研究所, 上海 200241

收稿日期:2020-05-06 出版日期:2020-12-25 发布日期:2020-12-23
通讯作者: 王少辉,主要从事畜禽细菌性传染病研究,Tel:021-34293412,E-mail:shwang0827@126.com;于圣青,主要从事畜禽细菌性传染病研究,Tel:021-34293461,E-mail:yus@shvri.ac.cn
作者简介:张耀东(1994-),男,河南新密人,硕士生,主要从事畜禽细菌性传染病研究,E-mail:837549574@qq.com
基金资助:
国家重点研发计划项目（2016YFD0500800；2018YFE0102200）；上海浦江人才计划（2019PJD057）；兵团财政科技计划项目（2020AB025）；中央级公益性科研院所基本科研业务费（2020JB03）

Development and Application of Multiplex PCR Method for the Drug Resistance Genes Detection in Avian Pathogenic Escherichia coli

ZHANG Yaodong, ZHU Hong, AFAYIBO Dossêh Jean Apôtre, WANG Yao, YI Zhengfei, XIN Suhua, TAO Chenglin, LI Tao, QI Jingjing, TIAN Mingxing, DING Chan, YU Shengqing*, WANG Shaohui*

Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

Received:2020-05-06 Online:2020-12-25 Published:2020-12-23

摘要/Abstract

摘要： 本研究旨在建立β-内酰胺类、四环素类、氨基苷类、酰胺醇类、磺胺类抗菌药物耐药基因的多重PCR检测方法，用于耐药基因的快速检测。根据GenBank公布的上述5类抗菌药物的耐药基因序列，设计17对特异性引物。通过优化PCR体系和反应程序，建立4组耐药基因（cat+floR+tetB+tetC；dfrA12+sul2+sul1+bla_CTX-M+bal_TEM-1；aac3+aph3+aadA1+strB；tetA+cmlA+strA+sul3）的多重PCR反应体系。然后，检测多重PCR方法的特异性和敏感性。利用建立的多重PCR方法检测42株禽致病性大肠杆菌的耐药基因，同时，检测其耐药性，比较分析耐药基因和耐药表型之间的相关性。结果显示，建立的4组多重PCR体系可有效扩增出17个耐药基因片段，测序结果表明特异性较好。敏感性结果表明，4组多重PCR体系的菌液敏感性分别为10³、10⁴、10⁴和10⁵CFU。禽致病性大肠杆菌分离株的耐药基因多重PCR和单重PCR检测结果一致，其携带的耐药基因和耐药表型的符合率为92.86%。本研究建立的耐药基因多重PCR方法能简便、快速地检测常见的耐药基因，可用于耐药基因的传播、流行调查。

关键词: 耐药基因, 多重PCR, 检测

Abstract: To detect the drug resistance genes, the multiplex PCR method for drug resistance genes of β-lactams, tetracyclines, aminoglycosides, amphenicols, and sulfonamides were developed. Based on the sequences of drug resistance genes from GenBank, 17 pairs of specific primers were designed. Then, 4 multiple PCR assays were established through the optimization of PCR reaction conditions and primers concentrations (cat+floR+tetB+tetC; dfrA12+sul2+sul1+bla_CTX-M+bal_TEM-1; aac3+aph3+aadA1+strB; tetA+cmlA+strA+sul3). The sensitivity and specificity of these assays were determined. The multiplex PCR assays were used to detect the drug resistance genes of 42 avian pathogenic Escherichia coli (APEC). The drug sensitivity of these APEC strains were also determined, which were compared to the distributions of drug resistance genes in these strains. The results showed that the 17 drug resistance genes were effectively and specifically amplified in these 4 optimized multiplex PCR assays. The detection limits of the 4 multiplex PCR were 10³, 10⁴, 10⁴ and 10⁵CFU of bacteria, respectively. The established multiplex PCR assays are specific and rapid for the detection of drug resistance genes in APEC strains, which showed 92.86% coincident with the drug resistance for these strains. The developed 4 multiplex PCR are simple and rapid assays for drug resistance genes detection, which can be used for the epidemiologic study for drug resistance genes.

Key words: drug resistance gene, multiplex PCR, detection

中图分类号:

S852.612

张耀东, 朱红, AFAYIBO Dossêh Jean Apôtre, 王瑶, 易正飞, 信素华, 陶程琳, 李涛, 祁晶晶, 田明星, 丁铲, 于圣青, 王少辉. 常见耐药基因多重PCR方法的建立及用于禽致病性大肠杆菌耐药性监测[J]. 畜牧兽医学报, 2020, 51(12): 3193-3198.

ZHANG Yaodong, ZHU Hong, AFAYIBO Dossêh Jean Apôtre, WANG Yao, YI Zhengfei, XIN Suhua, TAO Chenglin, LI Tao, QI Jingjing, TIAN Mingxing, DING Chan, YU Shengqing, WANG Shaohui. Development and Application of Multiplex PCR Method for the Drug Resistance Genes Detection in Avian Pathogenic Escherichia coli[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3193-3198.

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常见耐药基因多重PCR方法的建立及用于禽致病性大肠杆菌耐药性监测

Development and Application of Multiplex PCR Method for the Drug Resistance Genes Detection in Avian Pathogenic Escherichia coli

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