Preliminary results obtained with differents DNA extraction methods for the application in the molecular platform for the rapid detection of rifampicin resistant isolates

IMPERIALE BELÉN; PONTINO MÓNICA; MORCILLO NORA

Cartagena

Workshop; Primera reunión del proyecto financiado por la Comisión Europea: “Development of a molecular platform for detection of rifampicin and fluoroquinolone resistance in Mycobacterium tuberculosis”.; 2005

Corporación Corpogen, Bogotá, Colombia.

Objective: To select the best method for DNA extraction to be used with the molecular platform. Materials and methods: DNA extraction methods: A: boiled in mQ water, B: Guanidine thiocianate, C: boiled in TE-Tween 20. Samples: were included decontaminated sputa with different bacillary load (+, ++, +++), early cultures on MGIT 960, and LJ cultures. Results: Total of positives: 41/54 (76%). Total of negative: 13/54 (24%) Conclusions: The best DNA extraction method for decontaminated sputa is Guanidine Tiocyante, and for the culture sample is the boiled in water. There is no difference between the MGIT first and second sampling. There is no difference between 1Mc Farland and 10-4 1Mc Farland. Not all the positive AFB smear sputa had amplification results, therefore and in order to enhance the method, the presence of possible PCR inhibitors in respiratory clinical samples should be explored.