Mycobacteriology Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria Agdemir Wale ´ria-Aleixo, Erna G. Kroon, Marco A.S. Campos, Maria E. Margutti–Pinto, Claudio A. Bonjardim, Paulo C.P. Ferreira* Laborato ´rio de Vı ´rus, Departamento de Microbiologia, Instituto de Cie ˆncias Biolo ´gicas, Universidade Federal de Minas Gerais, P.O. Box 486, 31270 –901, Belo Horizonte, Brazil Abstract We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 g of DNA template was used, although better results were obtained with 5 g and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Mycobacterium tuberculosis; MHMA; 16S rRNA; PCR; diagnostic 1. Introduction Tuberculosis (TB) and other mycobacterial illnesses re- main the number one cause of infectious disease-related deaths worldwide. Annual increases in death rates of 10% per year related in part to the HIV epidemic have been reported (Bloom and Murray, 1992), with approximately 3 million deaths per year in this decade (CDC, 1993). At the same time, an increasing number of infections with Myco- bacterium tuberculosis (MTB) causing an extrapulmonary disease is also seen in AIDS patients (Persing, 1991; Pitch- enik et al., 1984). Of even greater concern than the increase in absolute numbers of mycobacterial infections is the emer- gence of previously rare strains, collectively termed “my- cobacteria other than M. tuberculosis” (MOTT), mostly those belonging to the M. avium complex (MAC) (Falkin- ham, 1996). Moreover, of particular importance, is the emergence of multi drug-resistant MTB, which cause over 90% mortality in immunocompromised hosts (Dooley et al., 1992; Shankar et al., 1991). Consequently, rapid, sensitive, specific and inexpensive methods for the early diagnosis of tuberculosis are needed to stop the spread of this disease (Thorton et al. 1998). The conventional diagnosis of tuberculosis based on the gold standard, i.e., culture-grown organisms, is extremely sensitive (10 –100 organisms per ml present for growth) but is expensive and time-consuming (2– 8 weeks) because of the slow doubling rate (Sritharan and Barker, 1991) of acid-fast bacilli (AFB). Direct staining procedures are rapid, but lack both sensitivity (Drobniesky et al., 1994) and spec- ificity, because Legionella and Nocardia are stained as well (Rogall et al., 1990). In addition, a positive result in direct staining for AFB does not discriminate among Mycobacte- rium spp. (Schirm et al., 1995). By overcoming the inherent limitations of staining and culture techniques, genotypic identification methods based * Corresponding author. Tel.: +55-31-499-2539; fax: +55-31-443- 6482. E-mail address: paulocpf@mono.icb.ufmg.br (P.C.P. Ferreira). Diagnostic Microbiology and Infectious Disease 36 (2000) 225–235 0732-8893/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0732-8893(00)00112-7