Novel use of guanidinium isothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material

Nucleic acid amplification technologies offer great promise for the rapid., sensitive and specific diagnosis of tuberculosis. However, the isolation o f inhibitor-Cree DNA from biological specimens is a bottleneck of the PCR a ssay. Here we describe a simple method for the isolation of PCR-amplifiable DNA of Mycobacterium tuberculosis from all types of samples of pulmonary a nd extrapulmonary origin tested. Briefly, it involves concentration of the bacilli by high-speed centrifugation, removal of PCR inhibitors by a wash s olution containing guanidinium isothiocyanate and the release of bacterial DNA by heating in the presence of detergents and Chelex-100 resin. The enti re process is accomplished within similar to 3 h. The method has been valid ated oil 780 samples of human, bovine and guinea pig origin including sputu m, cerebrospinal fluid, Pulmonary fluids, pus, fine needle aspirate, tissue . blood and milk. (C) 2001 Federation of European Microbiological Societies , Published by Elsevier Science B.V. All rights reserved.