Development and Evaluation of a Real-Time FRET Probe Based Multiplex PCR Assay for the Detection of Prohibited Meat and Bone Meal in Cattle Feed and Feed Ingredients

Abstract

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.

Molecular techniques for detecting and typing of bacteria, advantages and application to foodborne pathogens isolated from ducks

“…Real-time PCR is a polymerase chain reaction process in which the target DNA is amplified and quantified simultaneously within a reaction. Real-time PCR employs specific primer set, one or two probes and/or fluorescent dye to improve detection signals (Rensen et al 2006; Dhanasekaran et al 2010; Shi et al 2010). In real-time PCR, the amplified DNA is detected in real time as the reaction progresses instead of at the reaction end.…”

Molecular methods for the detection and characterization of foodborne pathogens

“…In addition, real-time PCR is a quantitative method and is often used to determine the number of pathogens in various samples [29,30]. Four types of indicators have been used most frequently in real-time PCR methods for pathogen detection: TaqMan probes [31,32], molecular beacons [33,34], fluorescence resonance energy transfer (FRET) hybridization probes [35,36] and SYBR Green dyes [37][38][39]. In addition to the specific primer set, one or two probes are required in the former three real-time PCR methods to be coupled with fluorescent dye to improve the detection signals, which are used for the increase of detection specificity and for the design of multiplex detection methods [40][41][42].…”

Development of a Rapid Immunodiagnostic Test for Pork Components in Raw Beef and Chicken Meats: a Preliminary Study

“…These include, methods based on electrophoresis (Kim & Shelef, 1986), isoelectric focusing (Jaussen et al, 1990), chromatography (Saeed et al, 1989), enzyme-linked immunosorbent assay (ELISA) (Hsieh et al, 1996;Chen et al, 1998;Macedo-Silva Macedo-Silva et al, 2000;Ofori & Hsieh, 2007;Asensio et al, 2008), electronic nose and ), electronic nose and gas chromatography mass spectrometer with headspace analyzer (GCMS-HS) (Nurjuliana et al, 2011), also fouri-fourier transform infrared (FTIR) spectroscopy (Rohman et al, 2011). In addition to those methods, methods based on In addition to those methods, methods based on DNA technology such as DNA hybridization (Ebbehøj & Thomsen, 1991;Ballin et al, 2009), polymerase chain reaction (PCR) (Matsunaga et al, 1995;Rensen et al, 2006;Rojas et al, 2009;Ballin et al, 2009;Rojas et al, 2010) have been used for more than two decades. Furthermore Aida et al (2005Aida et al ( & 2007 have developed a method for species identification from pork and lard samples using PCR analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene.…”