Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis

ArticleinJournal of microbiological methods 78(3):339-43 · August 2009with15 Reads
DOI: 10.1016/j.mimet.2009.07.006 · Source: PubMed
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.
    • "DNA amplification was detected by visualizing the colour of the final reaction mix after addition of ZYBR green to naked eye and under the UV light. 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 [9]. 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. "
    Full-text · Article · Jan 2014
    • "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). In this paper, we optimized this type visual LAMP assay with one-step operation and evaluated the real-time turbidity, specificity and sensitivity of event-specific detection of GM rice TT51-1. "
    [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.
    Full-text · Article · Jan 2014
    • ", we used calcein as an intercalating dye for detecting the amplification. When adding the fluorescent dye to the reaction in the one-step assay, amplification results can be observed by naked eye as a consequence of color change. Therefore this system can minimize the risk of contamination in laboratory and eliminate the need for electrophoresis.(Zhu et al. 2009) Although visual observation of the turbidity was similar to judgment graph of Loopamp real-time turbidimeter and calcein fluorescence assay (56fg), but detection by electrophoresis was 2-fold more sensitive (0.56fg). Thus, Brucellosis diagnosis especially in resource-poor settings can rely on turbidity assay without need to complex ins"
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Jun 2013
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