Mycobacteriology Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates Maria J. Torres a* , Antonio Criado a , Maite Ruiz b , Ana C. Llanos b , Jose C. Palomares a , Javier Aznar a,b a Departamento de Microbiologı ´a, Unidad de Microbiologı ´a Molecular, Facultad de Medicina, Sevilla, Spain b Servicio de Microbiologı ´a, H.H.U.U. Virgen del Rocı ´o, Universidad de Sevilla, Sevilla, Spain Received 14 May 2002; accepted 4 October 2002 Abstract In this study we designed two pairs of probes for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis with real-time PCR procedures. One pair of probes spans the region between codon 510 and 528 of the rpoB gene, and the other one screens for mutation at the regulatory region of the inhA gene. We have evaluated these probes in combination with two other pairs of probes previously described to detect mutations in 20 susceptible and 53 unique resistant M. tuberculosis clinical isolates. We were able to detect nine different mutations affecting five codons of the rpoB gene, two different mutations at codon 315 of the katG gene and a nucleotide substitution (C209T) in the regulatory region of the inhA gene within two hours turnaround. © 2003 Elsevier Science Inc. All rights reserved. 1. Introduction The increasing prevalence of tuberculosis in many areas of the world, coupled with the rise in drug-resistant and multidrug-resistant (MDR) Mycobacterium tubercu- losis strains, presents a major threat to global health (Heyman et al., 1999). Molecular approaches to the diagnosis of disease have begun to have a major influence on current clinical man- agement of various disorders. One of the most useful ap- plications of molecular testing has been the detection of Mycobacterium tuberculosis (Hale et al., 2001). Nucleic acid amplification-based genotypic assays are potentially the most rapid and sensitive methods for the detection of drug resistance and are theoretically able to provide a same- day diagnosis from clinical samples. During the past 10 years, several molecular methods have been developed for direct detection, identification, and susceptibility testing of mycobacteria. These methods can potentially reduce the diagnostic time from weeks to days (Heyman et al., 1999; Soini and Musser, 2001). The molecular basis of drug resistance in M. tuberculosis is becoming clearer. More than 96% of all rifampin-resistant strains have mutations in a 81-bp “core region” of the rpoB gene (Kapur et al., 1994; Telenti et al., 1993), and 12-75% of isoniazid-resistant strains have been found to contain mutations either in codon 315 of the katG gene or the inhA ribosomal binding site (Banerjee et al., 1994; Telenti et al., 1997; Zhang et al., 1992). In previous works (Gonza ´lez et al., 1999; Torres et al., 2002) we determined the frequency of mutations associated to rifampin and isoniazid resistance in Seville, using differ- ent molecular methods in circa 100 resistant clinical unique isolates of M. tuberculosis. The aim of this study was to design and evaluate two new pairs of probes to be used in combination with a previous described pair of probes (Torres et al., 2000; Torres et al., 2001), for detection of rifampin and isoniazid resistance in M. tuberculosis. One of the new probes, designed to detect rifampin resistance, covers the region between 510 and 528 codons of the rpoB gene. The second one was designed to detect the nucleotide substitution C209T at the regulatory region of the inhA gene for isoniazid resistance. The assay was evaluated with a collection of 20 susceptible and 37 resistant M. tuberculosis clinical isolates from two univer- sity hospitals from Seville and 16 resistant strains obtained from the Mycobacterium Reference Laboratory for Anda- lucia, Co ´rdoba, Spain. * Corresponding author. Tel.: +34-54557448; fax: +34-54377413. E-mail address: mjtorres@us.es (M.J. Torres). Diagnostic Microbiology and Infectious Disease www.elsevier.com/locate/diagmicrobio 45 (2003) 207–212 0732-8893/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. S0732-8893(02)00521-7