Comparison of DNA Extraction Results from Staphylococcus aureus Using the Boiling Method and the Combined BoilingCentrifugation-Bead Method in Terms of DNA Quality for PCR Testing
Keywords:
DNA Extraction, Staphylococcus aureus, PCRAbstract
DNA extraction is a critical step in molecular testing based on the Polymerase Chain Reaction (PCR). In Gram-positive bacteria such as Staphylococcus aureus, the DNA extraction process requires an effective lysis method due to their thick peptidoglycan layer. The boiling method is simple and economical; however, further development of extraction methods is still needed to improve the quality of the resulting DNA. This study aimed to compare the results of DNA extraction using the boiling method and the combined boilingcentrifugation-bead method in terms of the quality of Staphylococcus aureus DNA for PCR testing. This was a laboratory-based experimental study with a comparative design using 30 samples of Staphylococcus aureus bacterial suspensions standardized to a McFarland turbidity of 1.5. Fifteen samples were extracted using the boiling method, and 15 samples were extracted using the combined boiling-centrifugation-bead method. DNA quality was measured using a NanoDrop, including DNA concentration, DNA purity as determined by the A260/A280 ratio, and the A260/A230 ratio. The DNA’s ability to serve as a template was also evaluated through PCR amplification of the nuc gene. Data analysis was performed using the Independent Samples t-test and the Mann–Whitney U test. The boiling method yielded a mean DNA concentration of 12.57 ng/µL, a mean A260/A280 ratio of 2.2433, and a median A260/A230 ratio of -2.13. The combined boiling-centrifugation-bead method yielded a mean DNA concentration of 50.19 ng/µL, a mean A260/A280 ratio of 1.4220, and a median A260/A230 ratio of 0.89. Statistical analysis revealed significant differences in DNA concentration, DNA purity as indicated by the A260/A280 ratio, and the A260/A230 ratio between the two extraction methods (p<0.001). PCR results showed the formation of a target DNA band from the nuc gene measuring approximately 423 bp using both DNA extraction methods. The combined boiling-centrifugation-bead method yielded a higher DNA concentration and a better A260/A230 ratio compared to the boiling method.
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