Detection ofMycobacterium tuberculosisby Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples

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.

Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

“…To test for cross-hybridization using the AuNP probe with LAMP products from other pathogens, the amplicons from a VP AHPND DNA template (this study) served as the positive control for analysis by the AuNP colorimetric assay (analyzed by naked eye and confirmed by UV-vis spectrophotometry). The results were compared with those obtained using LAMP or RT-LAMP amplicons obtained by comparable LAMP methods for the following non-shrimp pathogens Mycobacterium tuberculosis (TB) [ 21 ] and Plasmodium (Malaria) [ 22 ], and for the shrimp pathogens WSSV [ 23 ], YHV [ 12 ], IMNV [ 13 ], IHHNV [ 24 ], TSV [ 25 ], LSNV [ 26 ] and PemoNPV [ 27 ].…”

Assessing the presence of Wuchereria bancrofti in vector and human populations from urban communities in Conakry, Guinea

“…In addition to high sensitivity, the LAMP is also less prone to the presence of irrelevant DNA and inhibition compared to PCR and has the advantage of shorter reaction time, simple readout system and the use of cheaper technology tools [ 34 – 36 ]. As such, it enjoys increasing popularity for the diagnosis of a host of diseases [ 22 , 37 , 38 ]. Indeed, LAMP represents a powerful tool that can be employed in the evaluation of LF control activities, especially in the end-game when interruption of LF transmission must be certified.…”

Diagnostic test accuracy of loop-mediated isothermal amplification assay for Mycobacterium tuberculosis: systematic review and meta-analysis

“…Although these studies have revealed generally very good diagnostic performance, there are considerable discrepancies between their results891011121314151617181920212223242526272829303132. In addition, none of the studies could describe precise diagnostic accuracy because of their limited statistical power.…”