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: Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was combined with a vertical flow (VF) nucleic acid detection strip to develop a universal assay for the detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). The Loop primers were labeled separately with biotin and fluorescein isothiocyanate (FITC) in this assay. Using optimized parameters, the whole reaction could be completed in <50min in a completely enclosed environment. The detection limit of this assay was found to be 1pg RNA, 30 tissue culture infective dose 50 (TCID50) virus, or 230 copies of recombinant plasmid DNA, which is relatively higher than that of RT-LAMP analyzed by agarose gel, RT-LAMP visualized by calcein, and the conventional RT-polymerase chain reaction (PCR). No false-positive results were obtained in the specificity assay. The efficiency of the RT-LAMP method was tested by analyzing 43 clinical samples, and the results were compared with those obtained by RT-PCR analysis, with the respective positive rates of 32.56% and 27.91%. This result confirmed that the method described is a rapid, accurate, and sensitive method for universal type II PRRSV detection. Also, this method can be used for the rapid detection of type II PRRSV during the early phase of an outbreak, especially for rapid veterinary diagnosis on the spot and in rural areas.Journal of virological methods. 09/2014;
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ABSTRACT: The global control of tuberculosis remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment, because treatment can only be initiated when infection is detected, and is guided by the results of antimicrobial susceptibility testing. To a large extent, non-molecular, immunological, and other biochemical methods are refinements or modifications of conventional methods, with the primary goal of providing more rapid test results. In contrast, molecular methods use novel technologies to detect the presence of Mycobacterium tuberculosis complex and genes conferring drug resistance. As a group, molecular technologies offer the greatest potential for laboratories in resource-rich countries because they have the highest sensitivity and specificity. In resource-poor settings, continued development of affordable, sensitive, and specific diagnostic tools will be required, where the incidence of disease is highest.European Journal of Clinical Microbiology 01/2015; · 2.54 Impact Factor
- Food Science and Technology Research 01/2014; 20(1):71-77. · 0.47 Impact Factor