多黏菌素类抗生素耐药性编码基因检测用质粒标准样品研制
作者:
作者单位:

1.西北农林科技大学食品科学与工程学院,陕西 杨凌 712100;2.杨凌质量技术检测检验所, 陕西 杨凌 712100;3.河北省林草花卉质量检验检测中心,河北 石家庄 050081;4.汉中市汉台区市场监督管理局,过街楼蔬菜批发市场监管所,陕西 汉中 723000;5.中国检验检疫科学研究院,北京 100176;6.中国食品药品检定研究院,北京 100500

作者简介:

陈进 女 在读研究生 研究方向为食品科学 E-mail: cxhchenjin@126.com

通讯作者:

杨保伟 男 教授 研究方向为食品科学 E­mail: ybwsheng@nwsuaf.edu.cn

中图分类号:

R155

基金项目:

“十三五”国家重点研发计划重点专项(2017YFC1601400)


Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection
Author:
Affiliation:

1.College of Food Science and Engineering, Northwest A&F University, Shaanxi Yangling 712100, China;2.Yangling Quality and Technical Inspection Institute, Shaanxi Yangling 712100, China;3.Hebei Forest, Grass and Flower Quality Inspection and Testing Center, Hebei Shijiazhuang 050081, China;4.Market Supervision and Administration Bureau of Hantai District of Hanzhong, Shaanxi Hanzhong 723000, China;5.Chinese Academy of Inspection and Quarantine, Beijing 100176, China;6.National Institutes for Food and Drug Control, Beijing 100500, China

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    摘要:

    目的 研制适合多黏菌素类抗生素耐药性编码基因检测用质粒DNA标准样品。方法 由National Center for Biotechnology Information(NCBI)数据库检索得到mcr-1mcr-3mcr-5基因参考序列,构建重组质粒和重组菌株;将重组菌传代至15代检测目标基因的遗传稳定性。提取重组质粒,真空干燥,制得标准样品。将标准样品水化、连续10倍梯度稀释,测定聚合酶链式反应(PCR)和实时荧光PCR(RT-qPCR)扩增目标基因的检出限。随机抽取质粒DNA标准样品,采用PCR法和紫外分光光度计法检验样品的均匀性及其在4 ℃、37 ℃、-20 ℃的贮存稳定性。结果 成功获得多黏菌素类抗生素耐药机制编码基因mcr-1mcr-3mcr-5基因片段,重组菌15代传代中目的基因遗传稳定。mcr-1mcr-3mcr-5标准样品PCR和RT-qPCR最低检出限分别为1.67×104 拷贝数/μL和1.67 拷贝数/μL、1.31×106 拷贝数/μL和13.1 拷贝数/μL、1.55×105 拷贝数/μL和1.55 拷贝数/μL。T检验表明,各基因随机抽取的12管标准样品质量间的F值均小于F临界值,且PCR均可检出,均匀性符合要求。标准样品在4 ℃贮存90 d、37 ℃贮存14 d、-20 ℃贮存360 d,定性、定量检验结果稳定、无极显著性差异。结论 本实验制备的mcr-1mcr-3mcr-5质粒DNA标准样品目的基因遗传稳定,均匀性和贮存稳定性良好。本研究结果可用于多黏菌素类抗生素耐药机制的快速预测和相关基因检测的质控样品。

    Abstract:

    Objective To develop plasmid DNA reference materials that can be used for polymyxin antibiotic resistance encoding genes rapid detection.Methods The reference sequence of mcr-1mcr-3 and mcr-5 were retrieved from the National Center for Biotechnology Information to construct recombinant plasmids and strains. The recombinant strains were subcultured for 15 generations to detect the genetic stability of the target gene. The recombinant plasmid was extracted and vacuum dried to prepare the standard sample. The limit of detection (LOD) of PCR and RT-qPCR was determined after the standard sample was hydrated and continuously diluted with 10-fold gradient, respectively. Multi-tube plasmid DNA standard samples were randomly selected to qualitatively and quantitatively evaluate the uniformity and storage stability at 4 ℃, 37 ℃ and -20 ℃ by PCR and UV spectrophotometer.Results Fragments of mcr-1mcr-3 and mcr-5 that encoding polymyxin resistance mechanism were successfully obtained. Target genes in the recombinant strains could stably inherited after subcultured for 15 generations. The LOD of PCR and RT-qPCR of mcr-1mcr-3 and mcr-5 standard samples were 1.67×104 and 1.67 copies/μL, 1.31×104 and 13.1 copies/μL, 1.55×105 and 1.55 copies/μL, respectively. The mass F values of 12 standard samples randomly selected for each gene were all less than the F critical value, each gene could be detected by RT-qPCR and indicating that the uniformity of the samples met the requirements. When standard samples were stored at 4 ℃ for 90 d, 37 ℃ for 14 d and -20 ℃ for 360 d, the qualitative and quantitative test result showed it was stable without significant difference.Conclusion In this study, plasmid DNA standard samples that carrying mcr-1mcr-3 and mcr-5 were prepared. The target genes could be stably inherited, the standard samples had good uniformity and storage stability, and could be used as a quality control samples for polymyxin antibiotics resistance encoding genes detection and mechanisms prediction.

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陈进,苏秀敏,杨静,曹彦卫,田园园,冯承谦,赵红阳,卢行安,崔生辉,杨保伟.多黏菌素类抗生素耐药性编码基因检测用质粒标准样品研制[J].中国食品卫生杂志,2022,34(2):203-210.

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  • 收稿日期:2021-10-19
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  • 在线发布日期: 2022-05-18
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