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                  • EI
                  • Scopus
                  • 食品科學與工程領域高質量科技期刊分級目錄第一方陣T1
                  • DOAJ
                  • EBSCO
                  • 北大核心期刊
                  • 中國核心學術期刊RCCSE
                  • JST China
                  • FSTA
                  • 中國精品科技期刊
                  • 中國農業核心期刊
                  • CA
                  • WJCI
                  • 中國科技核心期刊CSTPCD
                  • 中國生物醫學SinoMed
                  中國精品科技期刊2020
                  焦雪,董羽織,王靖雯,等. 基于RPA-LFD法的多黏菌素耐藥基因mcr-1可視化快速檢測方法建立與應用[J]. 食品工業科技,2023,44(18):209?216. doi: 10.13386/j.issn1002-0306.2022120034.
                  引用本文: 焦雪,董羽織,王靖雯,等. 基于RPA-LFD法的多黏菌素耐藥基因mcr-1可視化快速檢測方法建立與應用[J]. 食品工業科技,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法的多黏菌素耐藥基因mcr-1可視化快速檢測方法建立與應用

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

                  • 摘要: 目的:建立一種快速、高效、可視化的細菌多黏菌素耐藥基因mcr-1檢測方法,為其基層檢測的展開提供依據和便利。方法:利用重組酶聚合酶擴增結合膠體金側向流試紙條技術(Recombinase polymerase amplification combined with a lateral flow dipstick,RPA-LFD),輔以手持式膠體金讀數儀;根據mcr-1基因保守序列設計合成一對特異性RPA引物,通過對反應條件和體系的優化,以及特異性試驗、靈敏度試驗、模擬食樣試驗和實際樣品試驗,成功建立了可視化定量檢測細菌多黏菌素耐藥基因mcr-1的RPA-LFD方法。結果:在引物濃度400 nmol/L,引物比例1:1時,該方法最佳反應條件為Mg2+濃度14.0 mmol/L,反應溫度37 ℃,反應時間20 min;靈敏度好,標準曲線方程為y=0.117x+0.051,定量限為101~108 copies/μL,檢出限為101 copies/μL,比PCR法低一個數量級且模擬樣品檢出結果與PCR法一致。利用建立的RPA-LFD法對豬肉樣品、雞肉樣品、生豬養殖場環境樣品、肉雞養殖場環境樣品、大腸桿菌分離株和彎曲腸桿菌分離株各15份中多黏菌素耐藥基因mcr-1攜帶情況進行分析;RPA-LFD法與常規PCR法陽性樣本檢出率一致,共檢出9份mcr-1基因陽性樣品。RPA-LFD定量分析顯示,陽性樣品中mcr-1基因濃度在4.5×102~8.6×104 copies/μL之間。結論:本研究建立的細菌多黏菌素耐藥基因 mcr-1的RPA-LFD檢測法特異性強、靈敏性高、操作簡單,可廣泛應用于基層檢驗。

                     

                    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.

                     

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