Capture, Concentration, and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification

Abstract

We have previously investigated the extraction and concentration of bacteria from model systems using magnetic ionic liquid (MIL) solvents, while retaining their viability. Here, we combine MIL-based sample preparation with isothermal amplification and detection of Salmonella-specific DNA using Recombinase Polymerase Amplification (RPA). After initial developmental work with Serratia marcescens in water, Salmonella Typhimurium ATCC 14028 was inoculated in water, 2% milk, almond milk or liquid egg samples and extracted using one of two MILs, including: trihexyl(tetradecyl)phosphonium cobalt(II) hexafluoroacetylacetonate ([P66614+][Co(hfacac)3-]) and trihexyl(tetradecyl)phosphonium nickel(II) hexafluoroacetylacetonate ([P66614+][Ni(hfacac)3-]). Viable cells were recovered from the MIL extraction phase after the addition of modified LB broth, followed by a 20 min isothermal RPA assay. Amplification was carried out using supersaturated sodium acetate heat packs and results compared to those using a conventional laboratory thermocycler set to a single temperature. Results were visualized using either gel electrophoresis or nucleic acid lateral flow immunoassay (NALFIA). The combined MIL-RPA approach enabled detection of Salmonella at levels as low as 103 CFU mL-1. MIL-based sample preparation required less than 5 min to capture and concentrate sufficient cells for detection using RPA, which (including NALFIA or gel-based analysis) required approximately 30-45 min. Our results suggest the utility of MILs for the rapid extraction and concentration of pathogenic microorganisms in food samples, providing a means for physical enrichment that is compatible with downstream analysis using RPA.

Current State of Development of Biosensors and Their Application in Foodborne Pathogen Detection

“…First, the limit of detection (LOD) of the most recently developed biosensor is exceptionally low. The LOD can be as low as 5-100 CFU/mL for bacterial pathogens or pico-nanogram levels for toxins (16,32,35,59). The low LOD allows the users to cut down the pre-enrichment time, hence reducing the total detection time.…”

Biosensors for rapid detection of Salmonella in food: A review

“…Nowadays, much effort has been focused on novel sample pretreatment methods that are potential to be integrated into biosensors, including IMS (Du et al., 2018), microfluidic separation (He et al., 2013; Liu et al., 2019; Srbova et al., 2018), electrophoresis (Nguyen, Nguyen, Bui, & Seo, 2019; Zhang, Luo, et al., 2018), acoustophoresis (Ngamsom, Lopez‐Martinez, et al., 2016), magnetophoresis (Ngamsom, Esfahani, et al., 2016), and magnetic ionic liquid‐based extraction (Hice, Clark, Anderson, & Brehm‐Stecher, 2019). Among them, IMS has been regarded as one of the most useful tools for selective capture, separation, and concentration of Salmonella prior to detection.…”

Rapid and simultaneous visual screening of SARS-CoV-2 and influenza virufses with customized isothermal amplification integrated lateral flow strip

“…The core and direct clinical proof for detecting viruses still focuses on the nucleic acid detection. As one of the most promising isothermal molecular amplification tools, recombinase polymerase amplification (RPA) shows appealing features of operation simplicity, detection rapidness, and high sensitivity for detecting DNA or RNA from various pathogens at a constant temperate with 30 min ( Ahn et al, 2018 ; Choi et al, 2016 ; Crannell et al, 2014 ; Hice et al, 2019 ; Khater et al, 2019 ; Lobato and O'Sullivan, 2018 ; Piepenburg et al, 2006 ; Yang et al, 2020 ). Lateral flow strip (LFS) is a paper-based device which permits the performance of simplicity, rapidness, portability, cost-effectiveness, specificity, sensitivity, and low limit of detection for on-site detecting trace analytes ( Chen et al, 2020 ; He et al, 2018 ; Liu et al, 2017 ; Qin et al, 2021 ; Wang et al, 2020b , 2020c ; Wu et al, 2019 ; Yao et al, 2016 ; You et al, 2017 ; Zheng et al, 2016 ).…”