Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens

Ralph R. Meer From the Department of Veterinary Science, University of Arizona, Tucson, AZ 85721.

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J. Glenn Songer From the Department of Veterinary Science, University of Arizona, Tucson, AZ 85721.

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Abstract

Objective

To develop a multiplex polymerase chain reaction (PCR) assay to detect the genes for the major toxins of Clostridium perfringens (cpa [α toxin], cpb [β toxin], etx [ε toxin], iA [ι i toxin], and cpe [enterotoxin]).

Sample Population

Cultures of C perfringens obtained from collections and diagnosticians throughout North America.

Procedure

PCR primers were derived from published sequences of the genes for the major toxins (the “typing” toxins and enterotoxin). The concentration of each primer was titrated in a PCR assay to allow concurrent amplification of multiple target sequences, and other parameters of the assay were optimized (including concentrations of other reagents and times and temperatures for denaturation of template, annealing of primers, and primer extension). Specificity of the assay was measured by comparing genotype with phenotype (where it was known).

Results

The genotype, determined by multiplex PCR assay, agreed with phenotype in 99% (86/87) of strains where phenotype had been determined. Applied to 361 isolates from domestic animals and human beings, 95% (n = 344) were type A, and 12.8% (n = 44) of these contained cpe. The remaining 5% (n = 17) of the isolates were type B (n = 1), type C (n = 11), type D (n = 2), or type E (n = 4).

Conclusion and Clinical Relevance

Previous studies have documented usefulness of PCR in genotyping C perfringens. The multiplex assay is as effective, but simpler, and may be a useful alternative to standard in vivo typing methods. Results of genotyping of field isolates suggested the need for further epidemiologic study of clostridial enteritis, particularly as this pertains to predominant etiologic toxin types, and documented the presence of the reportedly rare genotypes B and E. (Am J Vet Res 1997;58:702–705)

Abstract

Objective

To develop a multiplex polymerase chain reaction (PCR) assay to detect the genes for the major toxins of Clostridium perfringens (cpa [α toxin], cpb [β toxin], etx [ε toxin], iA [ι i toxin], and cpe [enterotoxin]).

Sample Population

Cultures of C perfringens obtained from collections and diagnosticians throughout North America.

Procedure

PCR primers were derived from published sequences of the genes for the major toxins (the “typing” toxins and enterotoxin). The concentration of each primer was titrated in a PCR assay to allow concurrent amplification of multiple target sequences, and other parameters of the assay were optimized (including concentrations of other reagents and times and temperatures for denaturation of template, annealing of primers, and primer extension). Specificity of the assay was measured by comparing genotype with phenotype (where it was known).

Results

The genotype, determined by multiplex PCR assay, agreed with phenotype in 99% (86/87) of strains where phenotype had been determined. Applied to 361 isolates from domestic animals and human beings, 95% (n = 344) were type A, and 12.8% (n = 44) of these contained cpe. The remaining 5% (n = 17) of the isolates were type B (n = 1), type C (n = 11), type D (n = 2), or type E (n = 4).

Conclusion and Clinical Relevance

Previous studies have documented usefulness of PCR in genotyping C perfringens. The multiplex assay is as effective, but simpler, and may be a useful alternative to standard in vivo typing methods. Results of genotyping of field isolates suggested the need for further epidemiologic study of clostridial enteritis, particularly as this pertains to predominant etiologic toxin types, and documented the presence of the reportedly rare genotypes B and E. (Am J Vet Res 1997;58:702–705)

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