Heteroduplex mobility assay for rapid, sensitive and specific detection ofmycobacteria

We report an improved method for the detection and identification of mycoba cteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA f or detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stran ded, heteroduplex and homoduplex bands in PAGE was observed among the Mycob acterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a sp ecific heteroduplexes formed among different Mycobacterium species. Other b acterium species were distinguished from Mycobacterium due to slow migratin g heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were dete cted when as little as 1 eta g of DNA template was used, although better re sults were obtained with 5 eta g and when PCR products of sample test and m ycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate t he feasibility of using MHMA to detect and identify mycobacteria, 15 clinic al sample patients were tested. All MTB-positive clinical samples were iden tified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microor ganism showing differences within the 16S rRNA as well as to the identifica tion of new and unrecognized bacterial species. (C) 2000 Elsevier Science I nc. All rights reserved.