Preparation of mycobacterial DNA from blood culture fluids by simple alkali wash and heat lysis method for PCR detection

Abstract

A sodium iodide-isopropanol (NI) method was compared with an alkali wash and heat lysis (AH) procedure for the preparation and extraction of DNA from BACTEC 13A blood culture fluid samples from AIDS patients for use in a PCR for the detection and identification of mycobacteria. The sensitivity and efficiency of the DNA extraction methods were assessed by a multiplex PCR which detected the members of the genus Mycobacterium 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 PCR amplified mycobacterial DNA prepared by the AH procedure from 40 acid-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 for extracting DNA from 13 acid-fast bacillus-positive BACTEC fluid samples for PCR analysis. The study shows that the AH procedure in combination with the multiplex PCR is a simple, specific, and sensitive method which can be used in the routine diagnostic laboratory to detect and identify different members of the genus Mycobacterium in blood culture fluid samples from AIDS patients.