4种市售水产品抗生素耐药基因和intⅠ1实时荧光定量PCR检测
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浙江省食品药品监管系统科技计划项目(2018001)


Detection of Antibiotic Resistance Genes and intI1 in 4 Commercially Aquatic Products Using Real-time Quantitative PCR Method
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    摘要:

    为掌握水产品中抗生素耐药基因(ARGs)和Ⅰ类整合子intⅠ1含量及分布,本研究建立了水产品常见抗生素耐药基因实时荧光定量PCR(qPCR)检测体系。以含目的基因tetA、sul2、blaPSE、cmlA、qnrS、aac(6')-Ib、intⅠ1的重组质粒为标准品,建立qPCR标准曲线,按照已建立的qPCR体系对市售沼虾、鲫鱼、黄颡鱼、大黄鱼中ARGs和intⅠ1进行定量。结果表明,6种ARGs的最低检出限在11.5~202拷贝/μL之间,blaPSE的最低检出限为6.6×104 拷贝/μL。沼虾、鲫鱼、黄颡鱼、大黄鱼4种样品都存在高丰度的blaPSE,相对丰度范围为1.17×10-4~5.96×10-3,aac(6')-Ib的相对丰度在4种样品中都较低,在2.38×10-8~6.71×10-7之间。该检测体系以样品中细菌总基因组为模板,可对水产品中6种常见ARGs同时定量检测,能够较客观地揭示市售水产样品中ARGs的种类和数量分布信息,对控制水产品中ARGs污染并保障水产食品安全意义深远。

    Abstract:

    In order to understand the content and distribution of antibiotic resistance genes (ARGs) and intI1 in aquatic products, a quantitative real-time PCR method was established. Quantitative real-time PCR method was set up using recombinant plasmid containing the target genes as templates. And antibiotic resistance genes including tetA, sul2, blaPSE, cmlA, qnrS, aac(6')-Ib and intI1 were quantify by established qPCR methods. Results indicated that all the detected genes could be detected in the commercial Macrobrachium, Carassius auratus, Pelteobagrus fulvidraco and Larimichthys crocea. The detection limit of blaPSE was 6.6×104 copies/μL, and the detection limits of other ARGs were between 11.5 and 202 copies/μL. Among the four kinds of aquatic products, the relative abundances of blaPSE were the highest, ranging from 1.17×10-4 to 5.96×10-3, followed by the relative abundances of aac(6')-Ib ranging from 2.38×10-8 to 6.71×10-7. The obtained detection system can quantify 6 ARGs and intI1 gene in aquatic products simultaneously with the total bacterial DNA from the samples. The result could reveal the contaminated ARGs type and quantity of the commercial aquatic products, which is significant for controlling the ARGs pollution in aquatic products and ensuring the safety of aquatic food.

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毛海萍,叶 繁,赵巧灵,金仁耀,吴佳佳,戴志远.4种市售水产品抗生素耐药基因和intⅠ1实时荧光定量PCR检测[J].中国食品学报,2021,21(10):247-254

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  • 在线发布日期: 2021-11-22
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