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

被引：0
|
作者：
Jiao X. [1 ]
Dong Y. [1 ]
Wang J. [1 ]
Lü C. [1 ]
Fang J. [1 ]
Jiang H. [1 ]
机构：
[1] Key Laboratory of Specialty Agri-products Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou
来源：
Science and Technology of Food Industry | 2023年 / 44卷 / 18期
关键词：
bacterial colistin resistance gene mcr-1; lateral flow dipstick; quantification; rapid detection; recombinase polymerase amplification; visualization;
D O I：
10.13386/j.issn1002-0306.2022120034
中图分类号：
学科分类号：
摘要：
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. © 2023 Chinese Journal of Geological Hazard and Control. All rights reserved.
引用
收藏
页码：209 / 216
页数：7
相关论文
共 37 条
  • [31] JIN B, MA B, LI J, Et al., Simultaneous detection of five food-borne pathogens using a mini automatic nucleic acid extractor combined with recombinase polymerase amplification and lateral flow immunoassay[J], Microorganisms, 10, 7, (2022)
  • [32] LIU H, CAO R, XU W, Et al., A cost-effective method for the rapid detection of chicken adulteration in meat using recombinase polymerase amplification combined with nucleic acid hybridization lateral flow strip[J], Journal of Food Composition and Analysis, 111, (2022)
  • [33] LAI F Y, CHANG K C, CHANG C S, Et al., Development of a rapid sex identification method for newborn pigeons using recombinase polymerase amplification and a lateral-flow dipstick on farm[J], Animals, 12, 21, (2022)
  • [34] HOLMES D S, QUIGLEY M., A rapid boiling method for the preparation of bacterial plasmids[J], Analytical Biochemistry, 114, 1, (1981)
  • [35] CHE Y L, CHEN Q Y, CHEN J R, Et al., Development and application of recombinase-aided amplification assay (RAA) for detecting polymyxin-resistant mcr-1 gene[J], Progress in Veterinary Medicine, 43, 8, (2022)
  • [36] YU L Z, TAO L Y, WEI X F., Research and application progress in visualized rpa-lfd nucleic acid detection technology[J], Laboratory Animal and Comparative Medicine, 41, 6, (2021)
  • [37] ZHANG S S, HE B, LI S G, Et al., Rapid detection of Streptococcus suis with visual RPA-LFD technology[J], Acta Veterinaria et Zootechnica Sinica, 53, 2, pp. 538-547, (2022)
1234