Research The goal of my research is to develop and validate automated, user-friendly, high-throughput nucleic acid-based detection systems for application in diagnostic Veterinary medical, environmental and food safety procedures. I believe this process will require interdisciplinary collaboration and corporate partnering. Past research efforts have been devoted to development and evaluation of detection systems that utilize polymerase chain reaction (PCR) amplification for detecting Eperythrozoon suis in pig blood and Bovine Respiratory Syncytial Virus (BRSV) from cattle nasal swabs and tissue cultures. We have also developed PCR detection and differentiation methods for Cryptosporidium spp. from cattle feces and water samples and used limited culturing and PCR amplification for detecting Salmonella ssp. and E. coli isolates, particularly O157:H7. The E. coli research has targeted the PCR detection of virulence-associated genes (eaeA, stx-I, and stx-II) in dog, pig, cattle, and horse tissues, feces, feeds, and in processed meats and environmental samples. We have also developed and evaluated a 5’ nuclease assay for detecting virulent Yersinia enterocolitica in raw meats and other food products. We have collaborated with Drs. Randy Phebus and Y.C. “Dan” Fung in the Department of Animal Sciences & Industry, College of Agriculture, and Dr. Jan Sargeant, Food Animal Health & Management Center, College of Veterinary Medicine, KSU in these efforts. Support for developing, evaluating and automating the E. coli O157:H7 PCR-based TaqMan™ Sequence Detection System and the Salmonella PCR TaqMan kit (Applied Biosystems) has come from Applied Biosystems, Foster City, CA. Research applications are currently being investigated with cattle and wildlife feces, water samples, ground beef and other processed meat and environmental samples, different DNA recovery processes applicable to automated formats, and LIMS technology. Current research efforts involve the comparison of standard culture/biochemical recovery and identification process of E. coli O157:H7 from cattle feces with a 5’ nuclease assay that utilizes a secondary enrichment step; validating a semi-nested PCR procedure for detecting virulent Leptospirae spp. in dog urine; developing real-time PCR procedures for the automated detected of Porcine Respiratory & Reproductive Syndrome (PRRS) virus from boar semen and blood (currently funded by the National Pork Board); and collaborative studies with Cepheid, Sunnyvale, CA. Patents Patent (US & worldwide) issued on September 27, 1994 and assigned Patent No. 5,350,672. Entitled: Specific DNA primers and method to use same to detect Eperythrozoon suis. Patent (US & worldwide) issued on June 13, 1995 and assigned Patent No. 5,424,189. Entitled: Bovine respiratory syncytial virus detection and primers. Patent (US & worldwide) issued on July 31, 2001 and assigned Patent No. 6,268,143 B1. Entitled: Automated High Throughput E. coli O157:H7 PCR Detection System and Uses Thereof. Selected Publications Renter, DG, JM
Sargeant, R Oberst,
M Samadpuor. 2004. A longitudinal survey of Escherichia coli O157
strains from range cattle environments. In press, J. Clin. Microbiol. Bohra LK, RD Oberst, RK Phebus, MP Hays, RL Green and JM Sargeant. 2001. Evaluation of 5’ nuclease based detection assays to detect Escherichia coli O157:H7 from food products. Journal of Rapid Methods and Automation in Microbiology 9:143-160. Feder I., J.C. Nietfeld, J. Galland, T. Yeary, J. Sargeant, R. Oberst, M.L. Tamplin, and J.B. Luchansky. 2001. Comparison of cultivation and PCR-hybridization for detection of Salmonella in porcine fecal and water samples. J. Clin. Microbiol. 39(7):2477-2484. Vishnubhatla, A., R.D. Oberst, D.Y.C. Fung, W. Wonglumsom, M. P. Hays, and T. G. Nagaraja. 2001. Evaluation of a 5´-nuclease (TaqMan®) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples. J. Food Prot. 64(3):355-360. Sargeant BJM, JR Gillespie, RD Oberst, RK Phebus, DR Hyatt, LK Bohra, and J. Galland. 2000. A longitudinal study of Escherichia coli O157:H7 on cow-calf farms in Kansas. Am. J. Vet. Res. 61:1375-1379. Vishnubhatla, A., DYC Fung, RD Oberst, MP Hays, TG Nagaraja, S Flood. 2000. Development of a rapid 5’ nuclease assay for the detection of pathogenic strains of Yersinia enterocolitica. Appl. Environ. Microbiol. Appl. Environ. Microbiol. 66:4131-4135. J.M. Sargeant, D.J. Hafer, J.R. Gillespie, R.D. Oberst, S.J.A. 1999. Flood. Prevalence of Escherichia coli O157:H7 in white-tailed deer sharing rangeland with cattle. J Am. Vet. Med. Assoc... 215:792-794. Oberst, RD, MP Hays, LK Bohra, RK Phebus, CT Yamashiro, C Paszko-Kolva, JM Sargeant, and JR Gillespie. 1998. PCR-based amplification and detection of Escherichia coli O157:H7 utilizing an internal fluorogenic probe and the 5’ nuclease (TaqMan®) assay. Appl. Environ. Microbiol. 64(9):3389-3396. Stone, GG, MM Chengappa, MP Hays, RD Oberst. 1998. Application of PCR-OLA for the detection of salmonellae in processed meat, poultry, fish, and pet foods. Rapid Methods & Automation in Microbiology 6:119-128. Narayanan, S, TG Nagaraja, J Staats, MM Chengappa, RD Oberst. 1998. Biochemical and biological characterizations and ribotyping of Actinomyces pyogenes and Actinomyces pyogenes-like organisms from liver abscesses in cattle. Vet. Microbiol. 61:289-303. Narayanan, S, TG Nagaraja, N Wallace, J Staats, MM Chengappa, RD Oberst. 1998. Biochemical and ribotypic comparison of Actinomyces pyogenes and A. pyogenes-like organisms from liver abscesses, ruminal wall, and ruminal contents of cattle. Am. J. Vet. Res. 59:271-276.
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