Development and evaluation of a loop-mediated isothermal amplification combined with au-nanoprobe assay for rapid detection of Mycobacterium tuberculosis.

Loop-mediated isothermal amplification (LAMP) has been proposed as an inexpensive and easy to perform assay for molecular diagnostics. We present a novel strategy for the detection of LAMP amplicons derived from Mycobacterium tuberculosis by the use of Au-nanoprobes. When applied to a total of 93 clinical specimens, the LAMP assay demonstrated sensitivity and specificity higher than that of polymerase chain reaction and culture. The Au-nanoprobe augmented LAMP test platform with its advantages of robust reagents and a simple colorimetric detection method can be adapted easily for the rapid detection of other infectious disease agents at a low cost.

Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples.

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.

Detection of non-amplified Mycobacterium tuberculosis genomic DNA using piezoelectric DNA-based biosensors.

Piezoelectric DNA-based biosensor technology was developed as a new method for detection of M. tuberculosis. This method consists of immobilizing a thiol-modified oligonucleotide probe on the gold electrode surface of a quartz crystal, using a self-assembled monolayer method. The advantage of this study is that a non-amplified genomic bacterial DNA target was used. Instead, the genomic DNA was digested by restriction enzyme to obtain DNA fragments containing the target sequence. The fabricated biosensor was evaluated through an examination of 200 samples. No cross hybridization were observed against M. avium complex and other microorganisms. This target DNA preparation, without PCR amplification, will reduce time, costs, and the tedious step of amplification.