Rapid species identification of Mycobacterium tuberculosis complex by PCR
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Abstract:
Objective To develop a new,rapid and easy method for species identification of Mycobacterium tuberculosis complex(MTBC).Methods Seven sets of primers(Mtbc1-7) for MTBC were used to amplify the genomic DNA from the reference strains and 96 clinical isolates.Results The specificities of 7 sets of primers were identified by PCR with 3 MTBC reference strains,9 non-mycobacterium reference strains and 27 non-tuberculosis mycobacterium(NTM) reference strains. The results showed that mycobacterium and non-mycobacterium were identified by Mtbc1;NTM and MTBC by Mtbc2;M.microti by Mtbc3;M.bovis and M.bovis BCG by Mtbc4;M.africanum subtypeI and M.tuberculosis by Mtbc5;M.bovis BCG by Mtbc4 and Mtbc6 together;M.canettii by Mtbc5 and Mtbc7 together.Of 96 clinical isolates,20 MOTT,3 M.microti,1 M.bovis,1 M.bovis BCG,3 M.africanum subtypeⅠ,1 M.caprae,3 M.canettii,64 M.africanum subtypeⅡ and M.tuberculosis were identified.Conclusion PCR is a rapid and effective method for the species identification of MTBC,and can be used for molecular epidemiology.Keywords:
Mycobacterium tuberculosis complex
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Objective To establish the methodology for identification of clinical isolates of Mycobacterium and to identify the distribution of Mycobacterium species in hospitalized patients with tuberculosis in Heilongjiang province.It would provide the basis for accurate diagnosis of infections with Mycobacterium tuberculosis (MTB) and non-tuberculous Mycobacterium (NTM) as well as for effective therapy.Methods Three hundred and thirty Mycobacterium isolates from 330 tuberculosis patients hospitalized and clinically diagnosed in Harbin Chest Hospital from May 2007 to December 2008 were collected.Genomic DNA from the isolates was extracted after inactivation of Mycobacterium.Molecular identification was carried out using PCR,PCR-restriction fragment length polymorphism (RFLP) and sequencing.Results Among 330 clinical isolates,328 were identified as MTB complex (MTBC),accounting for 99.4％ (328/330); 2 were NTM,accounting for 0.6％ (2/330).Among 328 MTBC isolates,326were MTB,one was Mycobacterium Africanum(M.African) and one was Mycobacterium microti(M,microti),accounting for 99.4％ (326/328),0.3％ (1/328) and 0.3％ (1/328),respectively.It was found that the homology between the two NTM isolates and Mycobacterium avium intracellulare (MAC)was 99％ and 93％,respectively,suggesting that the two NTM isolates were MAC.The homology between the two NTM isolates was 93％.Conclusions PCR plus PCR-RFLP and sequencing provides an ideal method for precise identification of Mycobacterium species.In the clinically diagnosed tuberculosis patients,the predominant Mycobacterium species is MTB,however M.African,M.microti as well as NTM are also found.
Key words:
Mycobacterium; Species identification; Polymerase chain reaction; Sequence alignment
Mycobacterium tuberculosis complex
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Mycobacterium tuberculosis complex
Mycobacterium kansasii
Mycobacterium fortuitum
Housekeeping gene
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Objective To establish and evaluate a multiplex PCR technique to rapid differentiate Mycobacterium tuberculosis complex(MTBC)from non-tuberculous mycobacteria(NTM).Methods Three pairs of oligo nucleotide primers were used in the multiplex PCR reaction.A 473bp DNA fragment encoding oxyR-ahpCintergenic region in MTBC,a 235bp and 136bp DNA fragment encoding variable rpoBgene region from MTBC and NTM,respectively,were amplified.The multiplex PCR was assessed in 6reference strains of MTBC,50reference strains of NTM,312clinical strains of MTBC and 300clinical strains of NTM.Results The multiplex PCR produced two DNA fragments at the size 473bp and 235bp for MTBC reference strains,and one DNA fragment with the size 136bp for NTM reference strains.Among 312 MTBC clinical samples,the sensitivity was 99.36%(310/312)and specificity was 99.32%(294/296).Among 300NTM clinical samples,the sensitivity and specificity were 98.00%(294/300)and 100.00%(310/310)respectively.Conclusion The multiplex PCR can differentiate MTBC and NTM efficiently,and might be a valuable technique for clinical use.
Mycobacterium tuberculosis complex
Multiplex
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ABSTRACT Background: Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) may or may not have same clinical presentations, but the treatment regimens are always different. Laboratory differentiation between MTBC and NTM by routine methods are time consuming and cumbersome to perform. We have evaluated the role of GenoType® Mycobacterium common mycobacteria/additional species (CM/AS) assay for differentiation between MTBC and different species of NTM in clinical isolates from tuberculosis (TB) cases. Materials and Methods: A total of 1080 clinical specimens were collected from January 2010 to June 2012. Diagnosis was performed by Ziehl-Neelsen staining followed by culture in BacT/ALERT 3D system (bioMerieux, France). A total of 219 culture positive clinical isolates (BacT/ALERT® MP cultures) were selected for differentiation by p-nitrobenzoic acid (PNB) sensitivity test as and BIO-LINE SD Ag MPT64 TB test considering as the gold standard test. Final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType® Mycobacterium CM/AS assay (Hain Lifescience, Nehren, Germany). Results: Out of 219 BacT/ALERT® MP culture positive isolates tested by PNB as 153 MTBC (69.9%) and by GenoType® Mycobacterium CM/AS assay as 159 (72.6%) MTBC and remaining 60 (27.4%) were considered as NTM species. The GenoType® Mycobacterium CM/AS assay was proved 99.3% sensitive and 98.3% specific for rapid differentiation of MTBC and NTM. The most common NTM species were; Mycobacterium fortuitum 20 (33.3%) among rapid growing mycobacteria and Mycobacterium intracellulare 11 (18.3%) among slow growing mycobacteria. Conclusion: The GenoType® Mycobacterium assay makes rapid and accurate identification of NTM species as compared with different phenotypic and molecular diagnostic tool and helps in management of infections caused by different mycobacteria.
Mycobacterium tuberculosis complex
Nontuberculous Mycobacteria
Mycobacterium fortuitum
Mycobacterium kansasii
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Purpose. The purpose of this study was to evaluate the usefulness of a new diagnostic multiplexed bead-based bioassay (Quantamatrix Multiplexed Assay Platform; QMAP) system with shape-encoded silica microparticles for the rapid and accurate detection and identification of 23 mycobacterial species/groups, including Mycobacterium tuberculosis complex (MTBC). Methodology. A total of 295 mycobacterial clinical isolates cultured from respiratory specimens were used for identification of MTBC and non-tuberculous mycobacteria (NTM) using the QMAP system and the results were confirmed with PCR-restriction fragment length polymorphism (RFLP) analysis of the rpoB gene, rpoB sequence analysis and PCR-reverse blot hybridization assay (REBA). Results/Key findings. The Mycobacterium genus-specific probe of the QMAP system was positive for all 46 Mycobacterium reference strains and negative for 59 non-Mycobacterium strains. Based on 295 liquid culture-positive samples, both the culture-based conventional identification method and the QMAP system identified each 212 and 81 isolates as MTB and NTM species. The concordance rates for the identification of NTM species between the QMAP system and molecular assays were 92.8 % (77/83), 97.6 % (81/83) and 100 % (83/83) for PCR-RFLP, the rpoB sequence analysis and PCR-REBA, respectively. Conclusion. The QMAP system yielded rapid, highly sensitive and specific results for the identification of MTBC and NTM and accurately discriminated between NTM species within 3 h.
Identification
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Mycobacterium tuberculosis complex
SYBR Green I
Melting curve analysis
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Objective
To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculosis.
Methods
Four genes including 16S rRNA, Rv0577, RD9 and mtbk_20680 were selected to establish the multiplex PCR method. Specific primers were designed and the reaction system and conditions were optimized. The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Mycobacterium africanum (M.africanum), Mycobacterium bovis (M.bovis), M. bovis BCG, seven common non-tuberculosis Mycobacterium (NTM), seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis (MTB) isolated from patients with tuberculosis in Gansu Province of China.
Results
The fragments of 16S rRNA, Rv0577, RD9 and mtbk_20680 genes were 543 bp, 786 bp, 369 bp and 231 bp in length, respectively. MTB strains of the Beijing genotype were positive for all of the four genes, while the non-Beijing genotype strains were negative for mtbk_20680. M. africanum, M. bovis and M. bovis BCG strains were negative for 16S rRNA and Rv0577. NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria. Among the 93 clinical MTB strains, 80 (86.02%) belonged to the Beijing genotype and the other 13 (13.98%) were non-Beijing genotype strains. The specificity of the multiplex PCR method was 100%.
Conclusions
The established multiplex PCR method could accurately distinguish Mycobacterium, Mycobacterium tuberculosis complex (MTBC), NTM, MTB, and Beijing and non-Beijing genotype MTB with high sensitivity and specificity.
Key words:
Multiplex PCR; Mycobacterium; Mycobacterium tuberculosis; Beijing genotype
Mycobacterium tuberculosis complex
Multiplex
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ABSTRACT The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates and 3 M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow the routine identification of M. tuberculosis complex members in the clinical laboratory.
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Nontuberculous Mycobacteria
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