Comparison of four real-time PCRs for Listeria monocytogenes
Abstract number: P1739
Hess D.L., Savelkoul P., De Boer E., Heuvelink A., Spanjaard L., Ang C.W.
Objectives: Real-time PCR is a widely used method for detection of microbial nucleic acid sequences. In the past years, several real-time PCR's have been developed for the detection of Listeria monocytogenes. The aim of this study was to assess the sensitivity of four real-time PCR's for L. monocytogenes strains from food and human origin.
Methods: We selected two primer-probe combinations from literature, based on the number of investigated strains, sensitivity, specificity and Genbank nucleotide BLAST results. The primer-probe combinations are published by Rodríguez-Lázaro et al. and Oravcová et al., targeting the hly gene and actA gene, respectively. These primer-probe combinations were compared with a primer-probe set used by the Food and Consumer Product Safety Authority (VWA, unpublished), targeting the iap gene, and a primer-probe set developed by Huijsdens et al., targeting the hly gene. All PCR's used FAM-labelled probes. 168 human clinical isolates from the Netherlands Reference Laboratory for Bacterial Meningitis (RBM), 111 food isolates from the Food and Consumer Product Safety Authority (VWA), 3 complete genome sequenced strains (ATCC BAA-679 (=EGDe), F2365, HCC23) and L. monocytogenes type strain ATCC 15313 were used to determine the sensitivity of the four real-time PCR's. Specificity was assessed using a total of 40 non-L. monocytogenes strains, including all other Listeria species.
Results: The Huijsdens PCR detected 76 out of a total of 182 L. monocytogenes isolates (41.8%). These results were in line with nucleotide BLAST results. The complete set of isolates was analyzed with the remaining three PCRs. The Rodríguez PCR detected 277 out of 279 (99.3%) of all isolates, the VWA PCR and Oravcová PCR both accurately identified 278 out of 279 (99.6%) of all isolates. The undetected isolates originated from four different human clinical cases, all phenotypically as well as AFLP and PFGE typed as L. monocytogenes. Preliminary results suggest that detection limits of the PCRs are comparable to previously published results (<100 CFU/ml in the VWA PCR). Specificity of all PCRs is 100%.
Conclusion: The Huijsdens PCR identifies less than half of all isolates tested, whereas the other real-time PCRs identify almost all isolates accurately. The considerable variation in sensitivity of real-time PCRs should be taken into account when selecting real-time L. monocytogenes PCR for diagnostic use.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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