PREPARATION OF MYCOBACTERIAL DNA FROM BLOOD CULTURE FLUIDS BY SIMPLE ALKALI WASH AND HEAT LYSIS METHOD FOR PCR DETECTION

A sodium iodide-isopropanol (NI) method was compared with an alkali wa sh and heat lysis (AH) procedure for the preparation and extraction of DNA from BACTEC 13A blood culture fluid samples from AIDS patients fo r use in a PCR for the detection and identification of mycobacteria. T he sensitivity and efficiency of the DNA extraction methods were asses sed by a multiplex PCR which detected the members of the genus Mycobac terium and differentiated between M. intracellulare, M. tuberculosis, and M. avium isolates with a limit of detection of between 0.28 pg (67 cells) and 120 pg (28,571 cells) of standard mycobacterial DNA. The P CR amplified mycobacterial DNA prepared by the AH procedure from 40 ac id-fast bacillus-positive blood cultures with growth index values of > 20 U but not from 48 blood cultures with growth index values of <21 U. The AH method was about 10 times more sensitive than the NI method fo r extracting DNA from 13 acid-fast bacillus-positive BACTEC fluid samp les for PCR analysis. The study shows that the AH procedure in combina tion with the multiplex PCR is a simple, specific, and sensitive metho d which can be used in the routine diagnostic laboratory to detect and identify different members of the genus Mycobacterium in blood cultur e fluid samples from AIDS patients.