• EI
  • Scopus
  • 食品科学与工程领域高质量科技期刊分级目录第一方阵T1
  • DOAJ
  • EBSCO
  • 北大核心期刊
  • 中国核心学术期刊RCCSE
  • JST China
  • FSTA
  • 中国精品科技期刊
  • 中国农业核心期刊
  • CA
  • WJCI
  • 中国科技核心期刊CSTPCD
  • 中国生物医学SinoMed
中国精品科技期刊2020
焦雪,董羽织,王靖雯,等. 基于RPA-LFD法的多黏菌素耐药基因mcr-1可视化快速检测方法建立与应用[J]. 食品工业科技,2023,44(18):209−216. doi: 10.13386/j.issn1002-0306.2022120034.
引用本文: 焦雪,董羽织,王靖雯,等. 基于RPA-LFD法的多黏菌素耐药基因mcr-1可视化快速检测方法建立与应用[J]. 食品工业科技,2023,44(18):209−216. doi: 10.13386/j.issn1002-0306.2022120034.
JIAO Xue, DONG Yuzhi, WANG Jingwen, et al. Establishment and Application of Rapid Detection Method for Polymyxin Resistance Gene mcr-1 Based on RPA-LFD Method[J]. Science and Technology of Food Industry, 2023, 44(18): 209−216. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022120034.
Citation: JIAO Xue, DONG Yuzhi, WANG Jingwen, et al. Establishment and Application of Rapid Detection Method for Polymyxin Resistance Gene mcr-1 Based on RPA-LFD Method[J]. Science and Technology of Food Industry, 2023, 44(18): 209−216. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022120034.

基于RPA-LFD法的多黏菌素耐药基因mcr-1可视化快速检测方法建立与应用

Establishment and Application of Rapid Detection Method for Polymyxin Resistance Gene mcr-1 Based on RPA-LFD Method

  • 摘要: 目的:建立一种快速、高效、可视化的细菌多黏菌素耐药基因mcr-1检测方法,为其基层检测的展开提供依据和便利。方法:利用重组酶聚合酶扩增结合胶体金侧向流试纸条技术(Recombinase polymerase amplification combined with a lateral flow dipstick,RPA-LFD),辅以手持式胶体金读数仪;根据mcr-1基因保守序列设计合成一对特异性RPA引物,通过对反应条件和体系的优化,以及特异性试验、灵敏度试验、模拟食样试验和实际样品试验,成功建立了可视化定量检测细菌多黏菌素耐药基因mcr-1的RPA-LFD方法。结果:在引物浓度400 nmol/L,引物比例1:1时,该方法最佳反应条件为Mg2+浓度14.0 mmol/L,反应温度37 ℃,反应时间20 min;灵敏度好,标准曲线方程为y=0.117x+0.051,定量限为101~108 copies/μL,检出限为101 copies/μL,比PCR法低一个数量级且模拟样品检出结果与PCR法一致。利用建立的RPA-LFD法对猪肉样品、鸡肉样品、生猪养殖场环境样品、肉鸡养殖场环境样品、大肠杆菌分离株和弯曲肠杆菌分离株各15份中多黏菌素耐药基因mcr-1携带情况进行分析;RPA-LFD法与常规PCR法阳性样本检出率一致,共检出9份mcr-1基因阳性样品。RPA-LFD定量分析显示,阳性样品中mcr-1基因浓度在4.5×102~8.6×104 copies/µL之间。结论:本研究建立的细菌多黏菌素耐药基因 mcr-1的RPA-LFD检测法特异性强、灵敏性高、操作简单,可广泛应用于基层检验。

     

    Abstract: Objective: To develop a rapid, efficient and visual method for the detection of bacterial colistin resistance gene mcr-1, so as to provide the basis and convenience for the development of its detection at the grassroots level. Methods: Using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay, supplemented by a hand-held colloidal gold reader. According to the conserved sequence of the mcr-1 gene, a pair of specific RPA primers were designed and synthesized. Through the optimization of the reaction conditions and system, as well as the specificity test, sensitivity test, simulated food sample test and actual sample test, the RPA-LFD assay for visual and quantitative detection of bacterial colistin resistance gene mcr-1 was successfully established. Results: When the primer concentration was 400 nmol/L and the primer ratio was 1:1, the optimal reaction conditions of this method are Mg2+ concentration 14.0 mmol/L, reaction temperature 37 ℃ and reaction time 20 min. The sensitivity was good, the standard curve equation was y=0.117x+0.051, the quantification limit was 101~108 copies/μL, and the detection limit was 101 copies/μL, which was an order of magnitude lower than the PCR method and the detection result of the simulated sample was consistent with the PCR method. Carrying status of colistin resistance gene mcr-1 in each 15 pork samples, chicken samples, pig farm environmental samples, broiler farm environmental samples, Escherichia coli isolates and Enterobacter campylobacter isolates were analyzed by the established RPA-LFD assay. The detection rate of positive samples by RPA-LFD assay was consistent with that of conventional PCR method, and a total of 9 mcr-1 gene positive samples were detected. RPA-LFD quantitative analysis showed that the concentration of mcr-1 gene in positive samples was between 4.5×102~8.6×104 copies/µL. Conclition: The RPA-LFD detection method of the bacterial colistin resistance gene mcr-1 established in this study had strong specificity, high sensitivity, and simple operation, and could be widely used in grassroots inspections.

     

/

返回文章
返回