Optimization of Polymerase Chain Reaction for Detection of Clostridium botulinum Type C and D in Bovine Samples

Abstract

Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth.

Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

“…A large number of studies have focused on the detection of C. botulinum bont/A, bont/B, bont/E, and bont/F genes, which are responsible for toxin production leading to human botulism (1-4, 11, 13, 14, 17, 18, 30, 45). Several studies have also reported on the detection of type C (bont/C) and type D (bont/D) genes by conventional PCR (9,15,19,24,42,48,49), while only a few such genes have been detected by real-time PCR (25,28,29). Real-time PCR technique presents the advantages of being highly specific and sensitive with no need for a post-PCR detection assay, in contrast to conventional PCR.…”

Animal Botulism Outcomes in the AniBioThreat Project

“…A large number of studies have focused on the detection of C. botulinum bont/A, B, E, and F genes responsible for toxin production leading to human botulism. 7,[25][26][27][28][29][30][31][32][33][34][35] There are also several reports on the detection of type C (bont/C) and type D (bont/D) genes by conventional PCR [36][37][38][39][40][41][42] and a few by real-time PCR. [43][44][45][46][47] Real-time PCR has the advantages of being highly specific and sensitive with no need of post-PCR processing, in contrast to conventional gel-based PCR.…”

Botulism

“…Existen otras pruebas alternativas que tienen similar sensibilidad y confiabilidad 104 , como las técnicas de radioinmunoensayo, inmunoprecipitación, hemaglutinación pasiva y ELISA 12,20,75,76 . También se han desarrollado varias metodologías de reacción en cadena de polimerasa (PCR) para optimizar el diagnóstico sin utilizar animales de laboratorio 25,40,48,77 . El advenimiento de las toxinas proteómicas en el diagnósti-co molecular del botulismo marca el inicio del fin de las limitaciones asociadas a la identificación de las neurotoxinas.…”