Development of 11-plex MOL-PCR assay for the rapid screening of samples for Shiga Toxin-producing Escherichia coli

Travis A. Woods, Heather M. Mendez, Sandy Ortega, Xiaorong Shi, David Marx, Jianfa Bai, Rodney A. Moxley, T. G. Nagaraja, Steven W. Graves, Alina Deshpande

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx1, stx2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

Original languageEnglish (US)
Article number92
JournalFrontiers in Cellular and Infection Microbiology
Volume6
Issue numberAUG
DOIs
StatePublished - Aug 31 2016

Fingerprint

Shiga-Toxigenic Escherichia coli
Oligonucleotides
Ligation
Polymerase Chain Reaction
Nucleic Acids
Public Health
Image Cytometry
Foodborne Diseases
Multiplex Polymerase Chain Reaction
DNA
Virulence Factors
Microspheres
Flow Cytometry
Health

Keywords

  • EHEC
  • MOL-PCR
  • Multiplex PCR
  • STEC
  • Shiga toxin

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Microbiology (medical)
  • Infectious Diseases

Cite this

Development of 11-plex MOL-PCR assay for the rapid screening of samples for Shiga Toxin-producing Escherichia coli. / Woods, Travis A.; Mendez, Heather M.; Ortega, Sandy; Shi, Xiaorong; Marx, David; Bai, Jianfa; Moxley, Rodney A.; G. Nagaraja, T.; Graves, Steven W.; Deshpande, Alina.

In: Frontiers in Cellular and Infection Microbiology, Vol. 6, No. AUG, 92, 31.08.2016.

Research output: Contribution to journalArticle

Woods, Travis A. ; Mendez, Heather M. ; Ortega, Sandy ; Shi, Xiaorong ; Marx, David ; Bai, Jianfa ; Moxley, Rodney A. ; G. Nagaraja, T. ; Graves, Steven W. ; Deshpande, Alina. / Development of 11-plex MOL-PCR assay for the rapid screening of samples for Shiga Toxin-producing Escherichia coli. In: Frontiers in Cellular and Infection Microbiology. 2016 ; Vol. 6, No. AUG.
@article{66a4e18a5abc4a139bee73ede347511e,
title = "Development of 11-plex MOL-PCR assay for the rapid screening of samples for Shiga Toxin-producing Escherichia coli",
abstract = "Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx1, stx2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90{\%} analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.",
keywords = "EHEC, MOL-PCR, Multiplex PCR, STEC, Shiga toxin",
author = "Woods, {Travis A.} and Mendez, {Heather M.} and Sandy Ortega and Xiaorong Shi and David Marx and Jianfa Bai and Moxley, {Rodney A.} and {G. Nagaraja}, T. and Graves, {Steven W.} and Alina Deshpande",
year = "2016",
month = "8",
day = "31",
doi = "10.3389/fcimb.2016.00092",
language = "English (US)",
volume = "6",
journal = "Frontiers in cellular and infection microbiology",
issn = "2235-2988",
publisher = "Frontiers Media S. A.",
number = "AUG",

}

TY - JOUR

T1 - Development of 11-plex MOL-PCR assay for the rapid screening of samples for Shiga Toxin-producing Escherichia coli

AU - Woods, Travis A.

AU - Mendez, Heather M.

AU - Ortega, Sandy

AU - Shi, Xiaorong

AU - Marx, David

AU - Bai, Jianfa

AU - Moxley, Rodney A.

AU - G. Nagaraja, T.

AU - Graves, Steven W.

AU - Deshpande, Alina

PY - 2016/8/31

Y1 - 2016/8/31

N2 - Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx1, stx2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

AB - Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx1, stx2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

KW - EHEC

KW - MOL-PCR

KW - Multiplex PCR

KW - STEC

KW - Shiga toxin

UR - http://www.scopus.com/inward/record.url?scp=84990038875&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84990038875&partnerID=8YFLogxK

U2 - 10.3389/fcimb.2016.00092

DO - 10.3389/fcimb.2016.00092

M3 - Article

C2 - 27630828

AN - SCOPUS:84990038875

VL - 6

JO - Frontiers in cellular and infection microbiology

JF - Frontiers in cellular and infection microbiology

SN - 2235-2988

IS - AUG

M1 - 92

ER -