RESUMEN
Mycobacterium avium paratuberculosis (MAP) is thought to be associated with Crohn's disease in humans. Since Johne's disease affects dairy and beef cattle, meat may be a possible route of transmission of MAP to humans. In this study, we compared a rapid multiplex real time PCR assay and conventional culture to detect MAP in ground beef. The real time PCR assay amplifies both an internal sequence of the IS900 gene and an internal control targeting the ruminant-specific mt-cyt-b gene, in order to control for any false negative results. The sensitivity of this multiplex real time PCR assay on ground beef is 10(1) CFU/g and the sensitivity of conventional culture at 10(3) CFU/g. Furthermore, we conducted a survey of 200 retail ground beef samples using this system and did not detect the presence of MAP.
Asunto(s)
Contaminación de Alimentos/análisis , Productos de la Carne/microbiología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Bovinos , Recuento de Colonia Microbiana/métodos , Seguridad de Productos para el Consumidor , Humanos , Paratuberculosis/microbiología , Paratuberculosis/transmisión , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A modified forensic DNA extraction and real-time fluorescent polymerase chain reaction assay has been evaluated for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting the IS900 gene sequence of MAP. DNA was successfully extracted from manure samples by utilizing the Whatman FTA card technology, which allows for simple processing and storage of samples at room temperature. The FTA cards were washed and subjected to a Chelex-100 incubation to remove any remaining polymerase chain reaction (PCR) inhibitors and to elute the DNA from the FTA card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Detection of MAP DNA from bovine fecal samples spiked with known concentrations of viable MAP cells was obtained. The detection limits of the assay was consistently found to be between 10(2) and 10(4) colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive assay results. This cost-efficient assay allows reporting of results as early as 4 h after fecal collection, which can be particularly useful in highthroughput herd screening.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Estiércol/microbiología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Paratuberculosis/diagnóstico , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Bovinos , Recuento de Colonia Microbiana , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Heces/microbiología , Fluorescencia , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , TemperaturaRESUMEN
A novel real-time fluorescent multiplex polymerase chain reaction (PCR) assay for detecting and discriminating between bovine, ovine, and caprine contaminates in cattle feed was developed that simultaneously performs quality control monitoring on both the DNA extraction process and the level of PCR inhibition in the final DNA extract in a single PCR run. The assay used a single set of primers and two sets of FRET probes targeting the ruminant-specific mitochondrial cytochrome b gene. An internal control PCR reaction targeting a region of the chloroplast RNA polymerase beta-subunit (rpobeta) gene, which is conserved among plants, was incorporated into the ruminant multiplex PCR reaction in order to both monitor the DNA extraction method and to test for the presence of PCR inhibitors. The detection limit for bovine and ovine contaminates was evaluated over a period of two sets of six trials on 15 different types of cattle feed and feed ingredients spiked with known concentrations of bovine meat and bone meal (BMBM) and lamb meat and bone meal (LMBM). The assay was able to detect 0.05% w/w BMBM contamination and 0.1% w/w LMBM contamination in all samples of cattle feed and feed ingredients tested.