Related Experiment Videos
Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens
1Department of Veterinary Science, University of Arizona, Tucson 85721, USA.
American Journal of Veterinary Research
|July 1, 1997
Summary
A new multiplex PCR assay accurately identifies Clostridium perfringens toxin genes. This method simplifies genotyping and reveals the prevalence of different toxin types in animal and human isolates.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Bacterial Pathogenesis
Background:
- Clostridium perfringens is an important veterinary and human pathogen.
- Accurate toxin genotyping is crucial for understanding disease epidemiology and pathogenesis.
- Existing typing methods can be cumbersome and time-consuming.
Purpose of the Study:
- To develop and validate a multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of major Clostridium perfringens toxin genes.
- To assess the performance of the multiplex PCR assay against established phenotypic methods.
- To determine the prevalence of different toxin types among C. perfringens isolates from North American sources.
Main Methods:
- Development of multiplex PCR primers targeting alpha (cpa), beta (cpb), epsilon (etx), iota (iA), and enterotoxin (cpe) genes.
- Optimization of PCR conditions, including primer concentrations, reagent amounts, and thermal cycling parameters.
- Validation of the assay by comparing PCR-based genotyping with phenotypic toxin profiles in 87 C. perfringens strains.
- Application of the validated assay to genotype 361 isolates from domestic animals and humans.
Main Results:
- The multiplex PCR assay demonstrated high accuracy, with 99% agreement between genotype and phenotype.
- Type A strains were predominant (95%), with 12.8% of these carrying the enterotoxin (cpe) gene.
- The assay identified less common types, including Type C (11 isolates), Type D (2 isolates), Type E (4 isolates), and Type B (1 isolate).
Conclusions:
- The developed multiplex PCR assay is a simple, effective, and potentially superior alternative to traditional in vivo typing methods for Clostridium perfringens.
- Genotyping results highlight the need for further epidemiological studies on clostridial enteritis, focusing on prevalent toxin types.
- The study confirmed the presence of reportedly rare genotypes (B and E) in field isolates, underscoring their potential epidemiological significance.