title: Novel use of guanidinium isothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material.
abstract: Nucleic acid amplification technologies offer great promise for the rapid, sensitive and specific diagnosis of tuberculosis. However, the isolation of inhibitor-free DNA from biological specimens is a bottleneck of the PCR assay. Here we describe a simple method for the isolation of PCR-amplifiable DNA of Mycobacterium tuberculosis from all types of samples of pulmonary and extrapulmonary origin tested. Briefly, it involves concentration of the bacilli by high-speed centrifugation, removal of PCR inhibitors by a wash solution containing guanidinium isothiocyanate and the release of bacterial DNA by heating in the presence of detergents and Chelex-100 resin. The entire process is accomplished within approximately 3 h. The method has been validated on 780 samples of human, bovine and guinea pig origin including sputum, cerebrospinal fluid, pulmonary fluids, pus, fine needle aspirate, tissue, blood and milk.
authors.affiliation: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi-110029, India.
authors.forename: S
authors.initials: S
authors.lastname: Chakravorty
authors.forename: J S
authors.initials: JS
authors.lastname: Tyagi
mesh_terms: D000818:Animals; D001826:Body Fluids / Q000737:chemistry / Q000382:microbiology; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D006168:Guinea Pigs; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology; D014380:Tuberculosis, Bovine; D014397:Tuberculosis, Pulmonary
chemical_list: D004269:DNA, Bacterial; D017879:Isothiocyanates; C041943:2,3,4,6-tetra-O-acetylglucopyranosylisothiocyanate