99A-36 |
|
S. MITRA, Dept. of Animal Science, Oklahoma State Univ., 101 Animal Science Bldg., Stillwater, OK 74078-6051 and P. M. Muriana, Oklahoma Food & Agricultural Products Research & Technology Ctr., Oklahoma State Univ., Dept. of Animal Science, 109 FAPC, Stillwater, OK 74078-6055. Listeria monocytogenes is an important foodborne pathogen that has been responsible for numerous foodborne outbreaks and deaths. It’s high incidence on raw meat products has allowed L. monocytogenes to infiltrate processing environments of ready-to-eat meat products. Rapid methods to detect L. monocytogenes will assist processors in reducing the time required for testing. Our objectives were to examine and optimize Amplifluor UniPrimerTm for specific PCR detection of Listeria monocytogenes from meat products (raw and processed) following primary and secondary enrichment. Primer target sequences for PCR optimization were primarily based on the listeriolysin hemolysin gene (hlyA) using an Opticon II realtime thermal cycler. Primers were determined from multiple sequence alignment of known hlyA sequences (GenBank) using the primer analysis program in Vector NTI Suite. Realtime PCR was performed after initial inoculation, and after primary and secondary enrichments of various raw/ground and processed meats inoculated at 100-106CFU/gm. In both raw and RTE meats we were able to detect L.monocytogenes from post enrichment samples having Listeria levels of at least 103 cfu/ml. No amplification was obtained from negative controls. UniPrimerTm realtime PCR was able to positively detect meat samples inoculated with as few as 1 cfu/25gm with a maximum detection time of 2-days depending on the need for secondary enrichment. When using 16s rRNA DNA target sequences, we obtained higher fluoresence readings at lower threshold cycles (Ct) due to the six copies of the 16s rRNA genes per Listeria genome. Recent advances in the application of fluorescent labels on primers have provided new prospects for ‘user-friendly’ rapid PCR analysis of target microorganisms. The significance of the UniprimerTm system over other fluorescent-based PCR methods is that it allows universal application of the same labelled primer for different PCR targets and the potential to multiplex different targets in the same reaction tube with minimal optimization requirements.
Session 99A, Biotechnology: General
|