[Influence of reaction optimization on the results of PCR amplification of Panton-Valentine leukocidin genes among Staphylococcus aureus isolates].
Panton-Valentine leukocidin (PVL) is an important virulence determinant of Staphylococcus aureus. Polymerase chain reaction (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 temperatures for each set to investigate the effect of optimization of PCR parameters on the amplification results. A total of 321 S. aureus clinical isolates (84.4% methicillin-resistant S. aureus, 76.9% nosocomial) were included to the study. Two different primer sets and two different annealing temperatures were applied for the amplification of PVL gene. For this purpose while luk-PV-1 and luk-PV-2 primers and 55 degrees C and 58 degrees C annealing temperatures were used to amplify the 433 bp region inhabiting the luk-S PV and luk-F PV genes, PVLup and PVLdn primers and 50 degrees C and 48 degrees C annealing temperatures were used to amplify the 1918 bp region inhabiting the same genes. luk-PV-1 and luk-PV-2 primers yielded amplicons at 55 degrees C in 50.2% (161/321) and at 58 degrees C in 1.6% (5/321) of the isolates. To discriminate the positive amplicons from the crossly amplified PCR products, restriction endonuclease analysis was performed and it was observed that the five amplicons generated by luk-PV-1 and luk-PV-2 primers at 58 degrees C were cut by BspH1 enzyme as expected for the positive amplicons. None of the isolates yielded amplicons by PVLup and PVLdn primers at 50 degrees C, however, only 1.6% of the isolates yielded amplicons at 48 degrees C. These isolates were the same with the ones that were PVL positive with luk-PV-1 and luk-PV-2 primers at 58 degrees C. These data revealed that only 1.6% of the study isolates were PVL positive. These results showed that inappropriate cycling conditions may lead to false-negative or false-positive results in PVL-gene amplification. Restriction endonuclease or sequence analysis may be used to differentiate crossly-amplified sequences from PVL-positive amplicons. We suggest using different primers and different annealing temperatures in PVL gene amplification before concluding on the study results. Using positive and negative control strains in each run and optimization of the study protocol not only according to the literature data but also due view to the laboratory experience seems critical for obtaining reliable data.
Karahan
Zeynep Ceren
ZC
Ankara Universitesi Tip Fakültesi, Mikrobiyoloji ve Klinik Mikrobiyoloji Anabilim Dali, Ankara. ckarahan@medicine.ankara.edu.tr
Dolapçi
Iştar
I
Tekeli
Alper
A
D001427:Bacterial Toxins / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D005098:Exotoxins / Q000235:genetics*; D005188:False Negative Reactions; D005189:False Positive Reactions; D006801:Humans; D007956:Leukocidins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015183:Restriction Mapping; D013211:Staphylococcus aureus / Q000235:genetics* / Q000472:pathogenicity; D013696:Temperature; D014774:Virulence / Q000235:genetics
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin