View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Digital Repository @ Iowa State University Food Science and Human Nutrition Publications Food Science and Human Nutrition 11-30-2018 Capture, Concentration and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification Stephanie A. Hice Iowa State University, [email protected] Kevin D. Clark Iowa State University Jared L. Anderson Iowa State University, [email protected] Byron F. Brehm-Stecher Iowa State University, [email protected] Follow this and additional works at: https://lib.dr.iastate.edu/fshn_ag_pubs Part of the Food Chemistry Commons, Food Processing Commons, Human and Clinical Nutrition Commons, Immunology and Infectious Disease Commons, and the Molecular, Genetic, and Biochemical Nutrition Commons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ fshn_ag_pubs/215. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Food Science and Human Nutrition at Iowa State University Digital Repository. It has been accepted for inclusion in Food Science and Human Nutrition Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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 ombc ined 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. Disciplines Food Chemistry | Food Processing | Food Science | Human and Clinical Nutrition | Immunology and Infectious Disease | Molecular, Genetic, and Biochemical Nutrition Comments This accepted article is published as Hice, S.A.,Clark, K.D., Anderson,J.L., Brehm-Stecher,B.F., Capture, Concentration and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification. Analytical Chemistry, 2018. DOI: 10.1021/acs.analchem.8b04751. Posted with permission. This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/fshn_ag_pubs/215 Subscriber access provided by Iowa State University | Library Article Capture, Concentration and Detection of Salmonella in Foods Using Magnetic Ionic Liquids and Recombinase Polymerase Amplification Stephanie A. Hice, Kevin D. Clark, Jared L. Anderson, and Byron F. Brehm-Stecher Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04751 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 4, 2018 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts. is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 14 Analytical Chemistry 1 2 3 4 5 6 7 Capture, Concentration and Detection of Salmonella in Foods Using 8 Magnetic Ionic Liquids and Recombinase Polymerase Amplification 9 10 Stephanie A. Hice1‡, Kevin D. Clark2†‡, Jared L. Anderson2*, and Byron F. Brehm-Stecher1* 11 12 Department of Food Science and Human Nutrition1, Department of Chemistry2, Iowa State University, Ames, Iowa 50011, 13 United States 14 15 ABSTRACT: We have previously investigated the extraction and concentration of bacteria from model systems using magnetic ionic 16 liquid (MIL) solvents, while retaining their viability. Here, we combine MIL-based sample preparation with isothermal amplification 17 and detection of Salmonella-specific DNA using Recombinase Polymerase Amplification (RPA). After initial developmental work 18 with Serratia marcescens in water, Salmonella Typhimurium ATCC 14028 was inoculated in water, 2% milk, almond milk or liquid 19 egg samples and extracted using one of two MILs, including: trihexyl(tetradecyl)phosphonium cobalt(II) hexafluoroacetylacetonate + – + – 20 ([P66614 ][Co(hfacac)3 ]) and trihexyl(tetradecyl)phosphonium nickel(II) hexafluoroacetylacetonate ([P66614 ][Ni(hfacac)3 ]). Viable 21 cells were recovered from the MIL extraction phase after the addition of modified LB broth, followed by a 20 min isothermal RPA 22 assay. Amplification was carried out using supersaturated sodium acetate heat packs and results compared to those using a 23 conventional laboratory thermocycler set to a single temperature. Results were visualized using either gel electrophoresis or nucleic 24 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 25 RPA, which (including NALFIA or gel-based analysis) required approximately 30 - 45 min. Our results suggest the utility of MILs 26 for the rapid extraction and concentration of pathogenic microorganisms in food samples, providing a means for physical enrichment 27 that is compatible with downstream analysis using RPA. 28 29 30 31 Introduction: Salmonella is a ubiquitous, gram-negative to denature and separate duplex DNA (dsDNA), RPA 32 bacterium that is widespread in the environment and is a accomplishes this at lower temperatures using an enzyme, 7 33 contaminant in various foods, food ingredients, and in industrial recombinase. While RPA maintains several similarities to food processing environments.1 Infection typically results from PCR, including exponential amplification of target sequences, 34 ingestion of tainted food products, including consumption of a major difference between the two detection methods is the 35 contaminated poultry, eggs and dairy products. The Centers for temperature profile used for amplification. RPA is an 36 Disease Control and Prevention (CDC) estimates that isothermal process, which obviates the need for a thermal 37 nontyphoidal Salmonella spp. are responsible for 1.2 million cycler, allowing use of simple, small and inexpensive heating 38 cases of illness, 19,000 hospitalizations and nearly 380 deaths devices. The optimal temperature of RPA has been reported to 39 in the United States annually, resulting in an economic burden be 37 – 42 °C, but amplification of specific products has been 40 greater than 3.4 billion USD.2 Therefore, rapid, streamlined and demonstrated at temperatures ranging from 25 – 45 °C.8 41 field-deployable methods for detection of Salmonella spp.