Systematic interpretation of molecular beacon PCR for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Detection of multi-drug resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes two or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative PCR (qPCR) is a novel approach that takes ≤ 2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader-dependent and limits its clinical use. The aim of this study was to develop an objective, reader independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from two regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: The Texas-Mexico border and Colombia. Using coded DNA specimens, mutations within an 81 bp hotspot region of rpoB were established by qPCR with five beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold (Ct) of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIFsusceptible and resistant bacteria. Only then, the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype, and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture, and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, © 2009 Elsevier Inc. All rights reserved *Corresponding author: 80 Fort Brown, SPH Bldg Brownsville, Texas 78520 blanca.i. restrepo@uth.tmc. edu Telephone: (956) 882-5172 Fax: (956) 882-5152. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript Diagn Microbiol Infect Dis. Author manuscript; available in PMC 2011 May 1. Published in final edited form as: Diagn Microbiol Infect Dis. 2010 May ; 67(1): 37–46. doi:10.1016/j.diagmicrobio. 2009.12. 007. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.