Skip to main content
OSTI.GOVU.S. Department of Energy Office of Scientific and Technical Information
U.S. Department of Energy
Office of Scientific and Technical Information
 

Real-time PCR assays that detect genes for botulinum neurotoxin A–G subtypes

Journal Article · · Frontiers in Microbiology
 [1];  [2];  [2];  [3]
  1. U.S. Food and Drug Administration (FDA), Silver Spring, MD (United States)
  2. Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
  3. Northern Arizona Univ., Flagstaff, AZ (United States)
+ Show Author Affiliations

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories.

Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
USDOE; U.S. Department of Homeland Security (DHS)
Grant/Contract Number:
89233218CNA000001
OSTI ID:
2433932
Report Number(s):
LA-UR--23-33842
Journal Information:
Frontiers in Microbiology, Journal Name: Frontiers in Microbiology Vol. 15; ISSN 1664-302X
Publisher:
Frontiers Research FoundationCopyright Statement
Country of Publication:
United States
Language:
English

References (45)

A general method applicable to the search for similarities in the amino acid sequence of two proteins journal March 1970
Detection of Clostridium botulinum in fish and environmental samples using polymerase chain reaction journal August 1996
Development of Enrichment Semi-nested PCR for Clostridium botulinum types A, B, E, and F and Its Application to Korean Environmental Samples journal December 2007
An innovative molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia based on the GeneDisc cycler journal March 2011
Towards an international standard for detection and typing botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples: A European ring trial study to evaluate a real-time PCR assay journal March 2011
Prevalent toxin types of Clostridium botulinum in South Korean cattle farms journal February 2022
A bovine botulism outbreak associated with a suspected cross-contamination from a poultry farm journal September 2017
The UCSC SARS-CoV-2 Genome Browser journal September 2020
Genetic Characterization of Clostridium botulinum Type A Containing Silent Type B Neurotoxin Gene Sequences journal May 1996
Investigation ofClostridium botulinumin commercial poultry farms in France between 2011 and 2013 journal September 2014
Development and Application of Real-Time PCR Assays to Detect Fragments of theClostridium botulinumTypes A, B, and E Neurotoxin Genes for Investigation of Human Foodborne and Infant Botulism journal December 2004
Genomic Characterization of Newly Completed Genomes of Botulinum Neurotoxin-Producing Species from Argentina, Australia, and Africa journal March 2020
The Type F6 Neurotoxin Gene Cluster Locus of Group II Clostridium botulinum Has Evolved by Successive Disruption of Two Different Ancestral Precursors journal May 2013
3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures journal January 2000
Improved assay-dependent searching of nucleic acid sequence databases journal May 2008
Clostridium argentinense sp. nov.: A Genetically Homogeneous Group Composed of All Strains of Clostridium botulinum Toxin Type G and Some Nontoxigenic Strains Previously Identified as Clostridium subterminale or Clostridium hastiforme journal October 1988
Rapport D'introduction Du President Du Souscomite Clostridium Pour L'unification De La Nomenclature Des Types Toxinogenes De C. botulinum journal June 1953
Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction journal January 1996
Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum , Clostridium baratii and Clostridium butyricum journal August 2009
Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples journal August 2009
Detection and Confirmation of Clostridium botulinum in Water Used for Cooling at a Plant Producing Low-Acid Canned Foods journal November 2010
Implications of Genome-Based Discrimination between Clostridium botulinum Group I and Clostridium sporogenes Strains for Bacterial Taxonomy journal June 2015
Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples journal April 2010
Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material journal December 2001
Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains journal November 2006
Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction journal September 1993
Genetic confirmation of identities of neurotoxigenic Clostridium baratii and Clostridium butyricum implicated as agents of infant botulism. journal January 1988
Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. journal January 1994
Botulinum Neurotoxin-Producing Bacteria. Isn’t It Time that We Called a Species a Species? journal September 2018
Confirmation of botulism diagnosis in Australian bird samples by ELISA and RT rtPCR journal May 2021
Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements journal April 2011
Universal and specific quantitative detection of botulinum neurotoxin genes journal January 2010
BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay journal January 2012
Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia journal March 2016
Investigations into an Outbreak of Botulism Caused byClostridium botulinumType C/D in Laying Hens journal June 2015
Advancements in Detection Approaches of Severe Acute Respiratory Syndrome Coronavirus 2 journal December 2022
The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements journal February 2021
Clostridium botulinum and Clostridium perfringens Occurrence in Kazakh Honey Samples journal January 2019
Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense journal July 2021
Navigating the Complexities Involving the Identification of Botulinum Neurotoxins (BoNTs) and the Taxonomy of BoNT-Producing Clostridia journal September 2023
Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature journal January 2017
Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays journal June 2015
Clostridium botulinum spores in Polish honey samples journal September 2018
Screening for Clostridium botulinum Type A, B, and E in Cooked Chilled Foods Containing Vegetables and Raw Material Using Polymerase Chain Reaction and Molecular Probes journal February 2001
Investigation of botulism in free-range ducks farming in the Mekong Delta, Vietnam journal January 2022

Figures / Tables (11)

TABLE 1(p. 3)table TABLE 1
TABLE 2(p. 4)table TABLE 2
TABLE 3: Part 1(p. 5)table TABLE 3: Part 1
TABLE 3: Part 2(p. 6)table TABLE 3: Part 2
TABLE 4(p. 7)table TABLE 4
TABLE 5(p. 10)table TABLE 5
TABLE 6: Part 1(p. 12)table TABLE 6: Part 1
TABLE 6: Part 2(p. 13)table TABLE 6: Part 2
FIGURE 1(p. 14)figure FIGURE 1
FIGURE 2(p. 15)figure FIGURE 2
TABLE 7(p. 16)table TABLE 7

Similar Records

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature
Journal Article · Sat Dec 31 23:00:00 EST 2016 · Toxins · OSTI ID:1628582
Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay
Journal Article · Fri Jul 21 00:00:00 EDT 2017 · Applied and Environmental Microbiology · OSTI ID:1626064
De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics
Journal Article · Tue Mar 06 23:00:00 EST 2012 · Analytical and Bioanalytical Chemistry · OSTI ID:1815820

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Clostridium botulinum
PCR
assay
clinical diagnostics application
detection
food testing
highly sensitive and specific assay
real-time PCR assay
serotype A-G assay
surveillance and outbreak investigation
toxin