Multiplex Real-Time PCR Assay Design for Pathogen Detection, Quantification and Speciation
Robert S. Tebbs, PhD, Staff Scientist, Applied Biosystems*
A multiplex real-time PCR assay was designed to identify 3 species of Vibrio and an internal positive control using a four-dye configuration. Multiple strains were sequenced to identify target sites. Several individual assays within the multiplex contain more than one primer or probe due to strain to strain polymorphisms. Vibrio cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at =25 genomic copies. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of an exclusion panel that included nearest neighbors. Each assay in the multiplex exhibited 100 ± 10% efficiency over a 5 log range. Multiplex real-time PCR can simplify pathogen detection and reduce costs since 3 species can be analyzed in a single reaction tube. *In collaboration with: P.M.Brzoska, S.Bit, M.R.Furtado, and O.V.Petrauskene
Multiplexed Diagnostic Assays for Viral Pathogens of Cattle and Swine
Max V. Rasmussen, PhD, Biomedical Scientist, Biosciences Defense Division, Lawrence Livermore National Laboratory
Diagnostic tests for animal pathogens support a healthy livestock industry and reliable food supply. Lawrence Livermore National Laboratory developed multiplexed assays that simultaneously discriminate nucleic acid sequences of foreign and endemic viral pathogens affecting cattle and swine in a high-throughput format. The porcine-disease assay detects foot-and-mouth disease, vesicular stomatitis, swine vesicular disease, vesicular exanthema of swine and porcine reproductive and respiratory syndrome. The bovine-disease assay detects foot-and-mouth disease, malignant catarrhal fever, rinderpest, bovine viral diarrhea, bluetongue, bovine papular stomatitis, psuedocowpox, infectious bovine rhinotracheitis, vesicular stomatitis and bovine herpes mammalitis. Ongoing DHS-funded research includes enhancing sensitivity to FMDV, supporting the USDA-APHIS assay validation process and development of a FMDV sub-typing microarray.
Challenges for Sample Preparation and Pathogen Detection: Perspectives from the Food Industry
Daniel R. DeMarco, PhD, Senior Research Microbiologist, DuPont Qualicon
Many promising new sensor technologies purport to have potential applications in foodborne pathogen detection. Often there is little real understanding of the complexities and issues involved. In contrast to air or clinical samples which are relatively simple, food matrix presents a host of unique challenges that must be overcome before adoption of any particular sensing technology will occur in the food industry. These include but are not limited to; sensitivity requirements, sample size issues, matrix diversity, and presence of assay specific inhibitors. In addition, the food industry is notoriously low cost and often low tech in its historically preferred approaches to pathogen detection. This talk will highlight specific examples of the challenges faced for pathogen detection in foods, discuss some history and background of past and currently in-use technologies, and discuss future trends.
Rapid and On-Demand Detection and Characterization of Staphylococci Causing Bloodstream Infections
Yi-Wei Tang, PhD, Associate Professor of Pathology and Medicine, Director, Molecular Infectious Diseases Laboratory, Vanderbilt University Medical Center
Phenotypic methods take several days for identification and antimicrobial susceptibility testing of staphylococcal isolates after gram-positive cocci in clusters (GPCC) are observed in positive blood cultures. We developed and validated a StaphPlex system for species-level identification of staphylococci, detection of genes encoding Panton-Valentine leukocidin (PVL), and antimicrobial resistance determinants of staphylococci. The StaphPlex system was compared to phenotypic methods for organism identification and antimicrobial resistance detection for positive blood culture specimens in which GPCC were observed. Among a total of 360 GPCC specimens, 273 (75.8%), 37 (10.3%), 37 (10.3%), 1 (0.3%), 3 (0.8%), and 9 (2.5%) were identified by StaphPlex as coagulase-negative Staphylococcus (CoNS), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), or mixed infections of CoNS and MRSA, CoNS and MSSA, or nonstaphylococci, respectively, with an overall accuracy of 91.7%. The 277 CoNS-containing specimens were further identified to the species level with an overall accuracy of 80.1% compared to a combined reference identification. High very major errors were noticed when detection of aacA, ermA, ermC, tetM, and tetK was used to predict in vitro antimicrobial resistance, but relatively few major errors were observed when the absence of these genes was used to predict susceptibility. The StaphPlex system demonstrated high sensitivity and specificity when used for staphylococcal cassette chromosome mec typing and PVL detection. StaphPlex provides simultaneous staphylococcal identification and detection of PVL and antimicrobial resistance determinants within 5 h, significantly shortening the time needed for phenotypic identification and antimicrobial susceptibility testing
Immunochemical Technologies for Replacement of Rodent Bioassays in Sensitive Detection of Toxins in Foods
John Mark Carter, PhD, Research Leader, Foodborne Contaminants Research Unit, Agricultural Research Service, US Department of Agriculture*
Rapid sensitive assays for biothreat toxins that can be used to detect intentionally contaminated foods are now typically performed via bioassay in live mice. While bioassay provides essential data on bioavailability, animal models are technically, fiscally, and ethically challenging. Through careful application of state-of-the-art techniques for immunization and screening, we created new monoclonal antibody reagents (MAb) specific for detection of botulinum neurotoxin (BoNT). These MAbs bind BoNT so tightly that, in a sandwich ELISA, they are more sensitive than the rodent bioassay. These reagents are also useful for sample preparation and production of portable tests for field use. Through a CRADA with Safeguard Biosystems Corp., we used these MAbs to develop a simple dipstick assay that can detect BoNT in food at levels well below the human oral LD50. We also used the new MAbs to develop sample preparation methods based on immunomagnetic beads. In liquefied food extracts these beads rapidly and irreversibly bind all toxin present in a large sample. Sequestering the beads with a magnet effectively concentrates the toxin into a small volume suitable for laboratory testing. While the toxin is still bound to the beads, we can detect its highly specific peptidase activity using a fluorescence (FRET) based substrate, for detection of sublethal amounts of BoNT in foods.
* In collaboration with: L.Cheng, X.He, R.Rasooly, L.Stanker, and D.Brandon, USDA |
