Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis.
ABSTRACT Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 degrees C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.
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ABSTRACT: OBJECTIVES: A fundamental to global tuberculosis (TB) control is timely and accurate diagnosis of infectious cases of the disease. Among various methods, techniques based on nucleic acid amplification are the ones with promising prospects. The present study evaluates the diagnostic value of the recently developed IS6110-based loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis complex (MTBC) in sputum specimens. METHODS: In this cross-sectional study (2008-2009), IS6110-LAMP was evaluated on 101 sputum specimens from 93 highly suspected TB patients and compared to Amplicor MTB test and in-house IS6110-PCR and -nested PCR assays. Culture results or clinical recovery following anti-TB therapy was considered as a reference to prove the TB cases. RESULTS: The overall sensitivity of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 89.6% (69/77 specimens; 95% confidence interval [CI], 80.5-95.4%), 76.6% (59/77 specimens; CI, 65.6-85.5%), 79.2% (61/77 specimens; CI, 68.5-87.6%) and 59.7% (46/77 specimens; CI, 47.9-70.8%). The specificity and positive predictive value (PPV) were 100% for all the tests, and the negative predictive value (NPV) of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 75%, 57.1%, 60%, and 43.6%. There was an excellent overall agreement between LAMP and nPCR (k 0.92), and between LAMP and Amplicor (k 0.88), in addition to a better tolerance of IS6110-LAMP to inhibitors present in clinical specimens. CONCLUSION: The better diagnostic performance of IS6110-LAMP compared to Amplicor (p=0.009), nPCR (p=0.013) and PCR (p<0.0001) besides its rapidity, simplicity, and cost-effectiveness makes it a valuable method for the detection of MTBC in clinical samples, particularly in resource-limited settings.The Journal of infection 03/2013; · 4.13 Impact Factor
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ABSTRACT: Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 % and the specificity was 100 % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.BioMed research international. 01/2013; 2013:926230.
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ABSTRACT: The aim of this study was designing a LAMP method for the rapid detection of Brucella and development of a sensitive quantitative-LAMP (Q-LAMP) assay for quantification of brucellosis. In this study for the LAMP detection of the causative agent of brucellosis, we used specifically designed primers to target the omp25 conserved gene of Brucella spp. The sensitivity of the LAMP method was evaluated by preparing serial 10-fold dilution of omp25 gene containing plasmid followed by performing the LAMP reaction. To improve the assay as a quantitative test, LAMP products in the serial dilution were evaluated by Loopamp real-time turbidimeter system and then standard curve were generated by plotting time threshold values against log of copy number.The assay specificity was evaluated using Brucella genomic DNA and a panel containing genomes of 11Gram-positive and Gram-negative organisms. The LAMP assay was highly specific and no amplification products were observed from the non-Brucella organisms. The test sensitivity for visual detection of turbidity or fluorescent color change and also agarose gel electrophoresis was 560ng and 5.6ng, respectively. The lower limit of detection was 17 copies of the gene that could be detected in 50 min. The results of this study indicated that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Brucella spp. that may improve diagnostic potential in clinical laboratories. The LAMP assay because of the simplicity and low cost can be preferred to other molecular methods in infectious diseases. This article is protected by copyright. All rights reserved.Journal of Applied Microbiology 06/2013; · 2.20 Impact Factor