DNA extraction methods from sputa

IMPERIALE BELÉN; MORCILLO NORA

El Calafate, Santa Cruz, Argentina

Workshop; Segunda reunión del proyecto financiado por la Comisión Europea: ?Development of a molecular platform for detection of rifampicin and fluoroquinolone resistance in Mycobacterium tuberculosis?; 2006

Malbrán, Hospital Dr. Cetrángolo

Objectives: a) to compare three different rapid methods for DNA extraction, b) To assess the PCR sensitivity as amplification technique. Materials and method: Thirty spiked sputa were included in the study. These sputa were processed with two different decontaminated methods: USP and NALC-NaOH. The decontaminated sputa were used for: AFB smear examination, culture on LJ and MGIT 960 media, and for DNA extraction. The three methods used for DNA exaction were: Am: BSA 0.2%, B: Guanidine Thiocianate, C: Chelex-100 resine. A 250 pb of the gen rpoB was used to verify the DNA extraction. Results: Positive PCR results after decontamination by NALC-NaOH and the different extraction methods (%):  Am: 85.2, B: 82.7, C: 86.6. Positive PCR results after decontamination by USP and the different extraction methods (%):  Am and B: 83.3, C: 86.6. Conclusion: -No significant differences between NALC-NaOH and USP in PCR results. -No significant differences among the three DNA extraction methods. -Average time for Mycobacteria recovery by USP was 3 days shorter than using NALC-  NaOH. --The same rate of contaminated cultures was observed with both methods. -The rpoB PCR was sensitive and specific. -USP gave good PCR and cultures results but was HIGLY TOXIC and very expensive.