Influence of PCR parameters on the prevalence of Panton-Valentine leukocidin positivity in Staphylococcus aureus isolates
Abstract number: P1965
Karahan Z.C., Dolapci I., Tekeli A.
Objectives: Panton-Valentine leukocidin (PVL) is an important virulence determinant of Staphylococcus aureus strains. PCR amplification of the genes encoding PVL is the most widely used method for determining PVL-positivity. In this study, we used two different primer sets and different annealing conditions for each set to demonstrate the effect of PCR components on PVL-PCR results.
Methods: 321 non-duplicate S. aureus strains and GRE-14 positive control strain were included in this study. PCR amplification of the PVL genes were performed by (luk-PV-1 and luk-PV-2) and (PVLup and PVLdn) primers by previously published methods. For the first primer pair PCR was performed at 55 °C and 58 °C, and for the second set of primer at 50°C and 48 °C with the same thermal cycler.
Results: The first primer pair gave 161 (50.16%) PVL-positive amplicons at 55 °C and 12 (7.45%) at 58 °C. The second primer set gave 0 (0%) and 5 (1.56%) PVL-positive amplicons at 50 °C and 48 °C respectively. The 161 PVL-positive amplicons were subjected to BspH1 restriction endonuclease analysis in order to confirm the accuracy of the amplified region, and 12 (7.45%) of the 161 isolates which were also found to be positive with the first set of primers at 58 °C gave the expected restriction patterns of 180, 151 and 102 bp. Two of the 149 uncut amplicons were selected randomly and subjected to bi-directional automated sequence analysis with the same primers used for amplification, and the sequence obtained was compared with the sequences deposited to the GenBank. We found that 95% of this sequence was 99% identical to the genes encoding some conserved proteins of Staphylococcus aureus.
Conclusion: Our results show that primer selection and cycling conditions may lead to false-negative or false-positive results (due to cross-priming of the primers) in PVL-gene amplification. Restriction endonuclease or sequence analysis may be used to differentiate crossly-amplified sequences from true PVL-positive amplicons.
Acknowledgements: This study was supported by the Research Fund of Ankara University (Project No: 20050809006HPD).
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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