Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars

Raghavendra Joshi; Harish Janagama; Hari P. Dwivedi; T. M.A. Senthil Kumar; Lee Ann Jaykus; Jeremy Schefers; Srinand Sreevatsan

doi:10.1016/j.mcp.2008.10.006

Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars

Raghavendra Joshi, Harish Janagama, Hari P. Dwivedi, T. M.A. Senthil Kumar, Lee Ann Jaykus, Jeremy Schefers, Srinand Sreevatsan

Veterinary Population Medicine

Research output: Contribution to journal › Article › peer-review

248 Scopus citations

Abstract

Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10-40 CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 10¹-10² CFU S. Typhimurium/9 mL rinsate, while in a recirculation format, detection limits were 10²-10³ CFU/25 mL rinsate. Reproducible detection at <10¹ S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.

Original language	English (US)
Pages (from-to)	20-28
Number of pages	9
Journal	Molecular and Cellular Probes
Volume	23
Issue number	1
DOIs	https://doi.org/10.1016/j.mcp.2008.10.006
State	Published - Feb 2009

Bibliographical note

Funding Information:
This work was supported by USDA-NRICAP grant – Food Safety Research and Response Network (FSRRN) and in part by AES grant and a Center for Animal Health and Food Safety matching grant from University of Minnesota. Authors thank Drs. Nagaraja and Vanessa Lopez for providing the Salmonella serovars used in this study. Authors acknowledge the University of Minnesota's Biomedical Genomics Center for sequencing, fragment analysis, Minnesota Supercomputing Institute for sequence analysis software and Proteomics facility for mass spectrometry.

Keywords

Aptamers
Detection
Food borne pathogens
Pre-analytical processing
Salmonella

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10.1016/j.mcp.2008.10.006

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title = "Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars",

abstract = "Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10-40 CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 101-102 CFU S. Typhimurium/9 mL rinsate, while in a recirculation format, detection limits were 102-103 CFU/25 mL rinsate. Reproducible detection at <101 S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.",

keywords = "Aptamers, Detection, Food borne pathogens, Pre-analytical processing, Salmonella",

author = "Raghavendra Joshi and Harish Janagama and Dwivedi, {Hari P.} and {Senthil Kumar}, {T. M.A.} and Jaykus, {Lee Ann} and Jeremy Schefers and Srinand Sreevatsan",

note = "Funding Information: This work was supported by USDA-NRICAP grant – Food Safety Research and Response Network (FSRRN) and in part by AES grant and a Center for Animal Health and Food Safety matching grant from University of Minnesota. Authors thank Drs. Nagaraja and Vanessa Lopez for providing the Salmonella serovars used in this study. Authors acknowledge the University of Minnesota's Biomedical Genomics Center for sequencing, fragment analysis, Minnesota Supercomputing Institute for sequence analysis software and Proteomics facility for mass spectrometry. ",

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doi = "10.1016/j.mcp.2008.10.006",

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AU - Senthil Kumar, T. M.A.

AU - Jaykus, Lee Ann

AU - Schefers, Jeremy

AU - Sreevatsan, Srinand

N1 - Funding Information: This work was supported by USDA-NRICAP grant – Food Safety Research and Response Network (FSRRN) and in part by AES grant and a Center for Animal Health and Food Safety matching grant from University of Minnesota. Authors thank Drs. Nagaraja and Vanessa Lopez for providing the Salmonella serovars used in this study. Authors acknowledge the University of Minnesota's Biomedical Genomics Center for sequencing, fragment analysis, Minnesota Supercomputing Institute for sequence analysis software and Proteomics facility for mass spectrometry.

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N2 - Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10-40 CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 101-102 CFU S. Typhimurium/9 mL rinsate, while in a recirculation format, detection limits were 102-103 CFU/25 mL rinsate. Reproducible detection at <101 S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.

AB - Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10-40 CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 101-102 CFU S. Typhimurium/9 mL rinsate, while in a recirculation format, detection limits were 102-103 CFU/25 mL rinsate. Reproducible detection at <101 S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.

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Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars

Abstract

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