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 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.
Chakravorty
S
S
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi-110029, India.
Tyagi
J S
JS
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
D004269:DNA, Bacterial; D017879:Isothiocyanates; C041943:2,3,4,6-tetra-O-acetylglucopyranosylisothiocyanate