DifferentialIdentification of Mycobacterium tuberculosis Complexand Nontuberculous Mycobacteria by Duplex PCR Assay Using the RNAPolymerase Gene( rpoB )

Abstract

A novel duplex PCR method that can amplify the 235-and 136-bp rpoB DNAs of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM), respectively, with two different sets of primers was used to differentially identify 44 reference strains and 379 clinical isolates of mycobacteria in a single-step assay. Showing 100% sensitivity and specificity, the duplex PCR method could clearly differentiate M. tuberculosis complex and NTM strains. In addition, restriction fragment length polymorphism analysis and direct sequencing of the amplicon of NTM could be used to supplement species identification.With the recent global resurgence of mycobacterial infections, especially of tuberculosis, attributed to increased human immunodeficiency virus infection, there is an increasing demand for rapid, sensitive, and specific diagnostic methods for the detection and identification of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) in a clinical setting (2,3,4). NTM infection can cause clinical problems, as its pathogenic potential and susceptibilities to antituberculosis treatments vary (22). In addition, mixed infections of M. tuberculosis and NTM have been reported (16). Therefore, it has become important to be able to differentiate between the two during the early stage of the diagnostic procedure.The diagnosis of mycobacterial infection is accomplished by culture-based identification. Primary culture of slowly growing mycobacteria, without using the BACTEC culture system, usually takes 4 to 6 weeks or longer (10). However, recent methodological advances in molecular biology have provided alternative rapid approaches, e.g., the PCR and PCR-linked methods. For the rapid detection or identification of M. tuberculosis, target genes specific to mycobacteria are used in a PCR (7,8,17,19).Because the incidence of NTM infection is increasing, any methods capable of simultaneously determining the presence of M. tuberculosis and/or NTM would be useful. For this purpose, multiplex PCR, which simultaneously uses two or three different genes, has been frequently used, as the technique can specifically detect and identify different species of the genus Mycobacterium (6, 15, 18) and differentiate members of the M. tuberculosis complex (6, 9) in the routine diagnostic laboratory by using Mycobacterium genus-and species-specific genes.However, some of these genes have been found to lack specificity for M. tuberculosis. In addition, IS6110 PCR has been reported to produce false-negative (23) and false-positive (11) results, and the mtp40 gene is not present in all M. tuberculosis strains (21). These reports suggest that the multiplex PCR targeting of these genes has associated problems.In the present study, we used a simplified multiplex PCR assay, basically a duplex PCR (DPCR) assay, to differentiate M. tuberculosis complex and NTM by using a single gene, the RNA polymerase ␤-subunit-encoding gene (rpoB). To demonstrate the efficiency and usefulness of the DPCR assay in this context, we used it to identify refere...

Rapid detection of Mycobacterium tuberculosis complex by real-time PCR in sputum samples and its use in the routine diagnosis in a reference laboratory

“…Many regions of the mycobacterial genome have been used in molecular diagnosis of TB, such as IS6110, 16S rRNA, hsp65, rpoB, sdaA, devR and mpt64 (Manjunath et al, 1991;Lachnik et al, 2002;Kim et al, 2004;Chakravorty et al, 2005;Negi et al, 2007;Hwang et al, 2013;Nimesh et al, 2013). mpt64 is found as a single copy in the genome of MTBC species (Lee et al, 1994), and has been used for both pulmonary and extrapulmonary TB diagnosis (Tan et al, 1995;Dar et al, 1998;Martins et al, 2000;Bhanu et al, 2005;Therese et al, 2005;Takahashi & Nakayama, 2006;Dil-Afroze et al, 2008;Kusum et al, 2012;Sethi et al, 2012).…”

La diversité insoupçonnée du monde microbien

“…La faible variabilité des séquences d'ARNr 16S rend également confuse la limite des genres et des espèces bactériennes. De nouveaux marqueurs phylogénétiques : gyrB (gyrase) [5], rpoB (ARN polymérase) [6], hsp60 (heat-shock protein) [7]… ont été proposés en association à l'ARNr 16S pour la caractérisation de certaines espèces et genres bactériens cultivables. Ils devront être évalués pour la détection des bactéries non cultivables.…”

“…Relancer la recherche de nouvelles molécules à haute valeur ajoutée Sachant que 70 % des antibiotiques actuels sont produits par moins de 1 % des bactéries cultivables actuellement réperto-riées 6 , l'accès à la microflore de différents écosystèmes repré-sente une ressource biologique potentiellement inépuisable. L'utilisation des micro-organismes non cultivables (non cultivés) pour le développement de nouveaux produits à haute valeur ajoutée commence à voir le jour.…”

Challenges of Tuberculosis in Iran

“…This mutation can be found in 96.1% of rifampicin resistant M. tuberculosis isolates and occurs in rifampicin resistancedetermining region with 81-bp lengths corresponding to 507 -533 amino acid residues (35). In an Iranian report, it was demonstrated that 28% of isolates were rifampicin resistant, which was still lower compared to many areas in Africa and Europe (51.9% -68.9%) (36), while in the United States, about 13% of isolates from new tuberculosis cases have been resistant to one or more of the first-line antituberculosis drugs and 1.6% of cases are resistant to both isoniazid and rifampicin, defined as multidrug-resistant tuberculosis (37). Although multidrug-resistant tuberculosis in the re-treatment patients varies from 30% to 80% in different regions of the world (38), a study in Iran showed a notable success in the treatment of multidrug-resistant tuberculosis (39).…”