Abstract: Polymerase Chain Reaction (PCR) has been widely used due to its high specificity, sensitivity and rapid turn-around time. However, inhibitory factors may be co-extracted with the target nucleic acid that will hinder the performance of PCR. The major difficulty with mycobacteria is achieving optimal cell lysis. Due to a genuine need for an accurate test for the diagnosis of tuberculosis a comparison of DNA extraction methods was conducted. DNA extraction methods for Mycobacterium tuberculosis were evaluated including Triton, Chelex, Nonidet, SDS/Lysozyme and Silica-based methods on bacterial cells and spiked sputum. DNA extracted from the above procedures was diluted from 10-1 to 10-7 for use as templates in the PCR titration assay. The PCR end point was at a dilution of 10-2 when DNA was extracted using the Triton and Chelex extraction methods. It was 10-3 for Nonidet and 10-4 for SDS/Lysozyme extraction methods. DNA extractions by silica-based method had PCR end point titrations at the 10-5 dilution. The sensitivity of extraction by silica-based is 1-10 cells. In the present study silica-based method is effective extraction method. The end point titrations are identical for bacterial cells and spiked sputum. Inhibition was not observed in PCR with DNA isolated from spiked sputum. Silica-based method required the least labor and completion time
کلید واژگان :PCR, Chelex, Nonidet, SDS,Lysozyme
ارزش ریالی : 600000 ریال
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