Dual-Probe Assay for Rapid Detection of Drug-Resistant Mycobacterium tuberculosis by Real-Time PCR

Abstract

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.

Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA

“…To our knowledge, there have been no other reports in which accurate copy numbers of M. tuberculosis DNA have been measured by using a real-time (TaqMan) PCR technique (1,10,14,16,22). Although many previous studies have used a conventional single-step real-time PCR assay to quantitatively detect various infectious pathogens in different clinical samples, few have been able to describe accurate copy numbers of the causative pathogens (3,4,5,6,9,15,19,21,23).…”

“…However, the nested PCR assay using CSF samples has yet to be widely used in TBM diagnosis, due to its laborious and time-consuming procedure, which carries a high risk of sample contamination (8,12,17,20). Recently, real-time PCR assays have been applied in routine diagnostic laboratory testing (1,10,14,16,22). In addition to conventional qualitative analysis, real-time PCR assays make it possible to perform accurate quantitative analyses with a high degree of reproducibility (3,4,5,6,9,15,19,21,23).…”

Primary clarithromycin resistance toHelicobacter pylori: Is this the main reason for triple therapy failure?

“…However, in the past decade, different polymerase chain reaction (PCR) based approaches have been developed as alternative tools [15] . These techniques allow assessment of mutations in the peptidyltransferase region encoded in domain V of the H. pylori 23S ribosomal RNA region that confers clarithromycin resistance [16,17] . Undeniably, both culture and PCR-based methods have both advantages and limitations [18] .…”

“Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System

“…Several new molecular methods detecting drug resistance SNPs have already been set up (2,3,7,18,35,37,41). Like GenoType MTBDRplus or GeneXpert MTB/Rif (Cepheid), the spoligoriftyping test allows rpoB gene RRDR mutation detection with high sensitivity and specificity, with the additional advantage of allowing lineage or sublineage identification in a single test and in just a few hours.…”