Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle

A. A. Gurjar, N. V. Hegde, B. C. Love, Bhushan M. Jayarao

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium associated with a wide variety of diseases in domestic animals and humans. We have developed dual-labeled fluorescence hybridization probe (TaqMan®)-based real-time multiplex PCR assay for detection of toxin genes alpha (cpa), beta (cpb), iota (ia), epsilon (etx), beta2 (cpb2) and enterotoxin (cpe) of C. perfringens directly from cattle feces. The assay was standardized using ATCC reference strains of C. perfringens producing alpha, beta, iota, epsilon and enterotoxin, respectively. The assay for detection of beta2 toxin gene was standardized using a field strain of C. perfringens producing beta2 toxin. The minimum detection limit for the real time PCR assay ranged from 5 to 70 pg of DNA for the six toxin genes. A total of 307 fecal samples collected from seven dairy herds in Pennsylvania were analyzed using the multiplex assay. The real-time PCR assay revealed that cpa, cpb, ia, etx, cpb2 and cpe were detected in 68 (28.2%), 6 (2.5%), 6 (2.5%), 4 (1.6%), 164 (68%) and 11 (4.5%) of 241 PCR positive samples, respectively. The findings of the study revealed that C. perfringens beta2 toxin producing strains were widely prevalent in lactating cows in Pennsylvania and they may play an important role in C. perfringens associated diarrheal diseases.

Original languageEnglish (US)
Pages (from-to)90-95
Number of pages6
JournalMolecular and Cellular Probes
Volume22
Issue number2
DOIs
StatePublished - Apr 1 2008

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Clostridium perfringens
Multiplex Polymerase Chain Reaction
Feces
Real-Time Polymerase Chain Reaction
Enterotoxins
Genes
Domestic Animals
Spores
Limit of Detection
Fluorescence
Bacteria
Polymerase Chain Reaction
DNA
Clostridium perfringens cpb2 protein

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

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title = "Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle",
abstract = "Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium associated with a wide variety of diseases in domestic animals and humans. We have developed dual-labeled fluorescence hybridization probe (TaqMan{\circledR})-based real-time multiplex PCR assay for detection of toxin genes alpha (cpa), beta (cpb), iota (ia), epsilon (etx), beta2 (cpb2) and enterotoxin (cpe) of C. perfringens directly from cattle feces. The assay was standardized using ATCC reference strains of C. perfringens producing alpha, beta, iota, epsilon and enterotoxin, respectively. The assay for detection of beta2 toxin gene was standardized using a field strain of C. perfringens producing beta2 toxin. The minimum detection limit for the real time PCR assay ranged from 5 to 70 pg of DNA for the six toxin genes. A total of 307 fecal samples collected from seven dairy herds in Pennsylvania were analyzed using the multiplex assay. The real-time PCR assay revealed that cpa, cpb, ia, etx, cpb2 and cpe were detected in 68 (28.2{\%}), 6 (2.5{\%}), 6 (2.5{\%}), 4 (1.6{\%}), 164 (68{\%}) and 11 (4.5{\%}) of 241 PCR positive samples, respectively. The findings of the study revealed that C. perfringens beta2 toxin producing strains were widely prevalent in lactating cows in Pennsylvania and they may play an important role in C. perfringens associated diarrheal diseases.",
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Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle. / Gurjar, A. A.; Hegde, N. V.; Love, B. C.; Jayarao, Bhushan M.

In: Molecular and Cellular Probes, Vol. 22, No. 2, 01.04.2008, p. 90-95.

Research output: Contribution to journalArticle

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T1 - Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle

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