Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/7/2002
Publication Date: 5/18/2003
Citation: Bayles, D.O. 2003. Development of a real-time multiplex pcr assay for detection of listeria monocytogenes and comparison of dual-labeled probes, molecular beacons, and sybrâ green i for detection in real time. 103rd General Meeting of the American Association for Microbiology. Paper No. 057. p. 499.
Technical Abstract: A PCR assay for Listeria and the species L. monocytogenes was designed and optimized for real-time detection with a Cepheid SmartCycler. One previously published primer was used along with three newly designed primers in order to detect the genus Listeria and the species L. monocytogenes. The genus amplicon was a 239-bp product produced from amplification of a 16S rDNA sequence, and the species amplicon was a 200-bp product produced from amplification of a L. monocytogenes specific hlyA DNA sequence. The time and temperature parameters for cycling were optimized using a response surface model designed and evaluated using the SAS Institute's ADX designed experiment interface. Analysis of the response surface model indicated optimal cycling conditions consisting of initial denaturing at 94.5°C for 2 min, followed by cycling conditions of denaturing at 96°C for 1 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s. Experimental results indicated that the interaction between annealing temperature and time, and the extension time were the most critical for optimizing the multiplex PCR on the Cepheid instrument. Fluorescent probes were added to the optimized reaction to evaluate the multiplex PCR in real-time using dual-labeled probes, molecular beacons, and SYBR Green I. Dual-labeled probes were found to produce reliable results with the fewest technical problems. Molecular beacons made to the same sequence as the dual labeled probes worked well for the species-specific amplicon, but were not effective for the genus amplicon. A response surface model generated to optimize reactions containing SYBR Green I indicated that SYBR Green I produced increased background and decreased amplification of the desired products. The final real-time multiplex PCR was checked for accuracy using UVM enrichment media and a variety of bacteria.