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.
"Surprisingly, this kind of nonspecific amplification has neither been recorded in other studies where LAMP has been validated to diagnose TB nor been recorded as a common flaw that could occur with LAMP technique - . Considering the area of the target region covered by the six primers used in the amplification of MTB by LAMP assay, which is about 95%, we suspect that this nonspecific amplification could be due to any kind of self amplification of the primers. "
"Recently, an improved LAMP strategy had been reported which applied the manganous ion and calcein, a fluorescent metal indicator, into the reaction system so that the result could be readily distinguished by colour change from orange to green without opening tube after amplification (Tomita et al., 2008). And this novel visual LAMP method had been successfully applied to Mycobacterium (Zhu et al., 2009) and Brucella (Pan et al., 2011). "
[Show abstract][Hide abstract] ABSTRACT: Loop-mediated isothermal amplification (LAMP), a novel DNA amplification technique, is widely used in molecular diagnosis although it is very easy to be contaminated by the huge amount of its own amplicons. An improved visual LAMP method for the event specific identification of GM rice TT51-1 and a rice endogenous reference gene (sucrose phosphate synthase, SPS) was established. Pre-loaded calcein and manganous ion into the LAMP reaction offered not only an easy way to read results by colour change from orange to green but also a closed-tube system to reduce contaminations caused by amplification products. The LAMP assays could be finished within 60 min, and the limits of detection (LODs) were estimated as low as 10 and 20 copies of rice haploid genomic DNA, respectively, about 10-fold more sensitive than that of conventional PCR. The specificities of the assays were also well evaluated with optimized system and conditions. The developed one-step visual LAMP assays in this study provided a promising tool for the convenient and effective detection of GM crops.
Food Science and Technology Research 01/2014; 20(1-1):71-77. DOI:10.3136/fstr.20.71 · 0.35 Impact Factor
"Referring to the test, no expensive equipment was required and the results could be determined within approximately 1 h (not including DNA preparation time). LAMP-LFD was faster than LAMP-gel electrophoresis (2 h 30 min) and much faster than PCR-gel electrophoresis (3 h 30 min) . "
[Show abstract][Hide abstract] 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.
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