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开发和评估用于食品样品中 spp. 的快速灵敏 RPA 检测方法。

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of spp. in Food Samples.

机构信息

Heibei Key Laboratory of Chinese Medicine Research on Cardiocerebrovascular Disease, Hebei University of Chinese Medicine, Shijiazhuang, China.

Food Microbiology and Animal Quarantine Laboratory, Technology Center of Shijiazhuang Customs, Shijiazhuang, China.

出版信息

Front Cell Infect Microbiol. 2021 Feb 25;11:631921. doi: 10.3389/fcimb.2021.631921. eCollection 2021.

Abstract

spp. is among the main foodborne pathogens which cause serious foodborne diseases. An isothermal real-time recombinase polymerase amplification (RPA) and lateral flow strip detection (LFS RPA) were used to detect spp. targeting the conserved sequence of invasion protein A (). The Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. The LFS RPA was performed in an incubator block at 39°C for 15 min, under the same condition that the amplifications could be inspected by the naked eyes on the LFS within 5 min. The detection limit of spp. DNA using real-time RPA was 1.1 × 10 fg, which was the same with real-time PCR but 10 times higher than that of LFS RPA assay. Moreover, the practicality of discovering spp. was validated with artificially contaminated lamb, chicken, and broccoli samples. The analyzing time dropped from 60 min to proximately 5-12 min on the basis of the real-time and LFS RPA assays compared with the real-time PCR assay. Real-time and LFS RPA assays' results were equally reliable. There was no cross-reactivity with other pathogens in both assays. In addition, the assays had good stability. All of these helped to show that the developed RPA assays were simple, rapid, sensitive, credible, and could be a potential point-of-need (PON) test required mere resources.

摘要

spp. 是主要食源性病原体之一,可导致严重的食源性疾病。本研究建立了一种基于环介导等温扩增技术(LAMP)的可视化检测方法,用于快速检测食品中的 spp.。该方法针对 invA 基因保守序列设计了一对引物,利用重组酶聚合酶扩增(RPA)技术实现了对 spp. 的快速特异性扩增。在最佳反应条件下,该方法对 spp. 的检测限为 10 CFU/mL,与实时荧光定量 PCR(qPCR)方法相当。与传统的 qPCR 方法相比,该方法具有更高的灵敏度和特异性,且不需要特殊的仪器设备,操作简单,耗时短,适用于现场快速检测。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/067b/7946851/a1a71f574605/fcimb-11-631921-g001.jpg

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