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.

[1]  X. Nassif,et al.  RAPID DIAGNOSIS OF TUBERCULOSIS BY AMPLIFICATION OF MYCOBACTERIAL DNA IN CLINICAL SAMPLES , 1989, The Lancet.

[2]  N. Arunrut,et al.  Rapid and sensitive detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. , 2011, Journal of virological methods.

[3]  N. Arunrut,et al.  Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateral-flow dipstick. , 2010, Molecular and cellular probes.

[4]  Kosum Chansiri,et al.  Detection of Non-Amplified Mycobacterium tuberculosis Genomic DNA Using Piezoelectric DNA-Based Biosensors , 2010, Sensors.

[5]  P. Reddi,et al.  Evaluation of PCR based test for the detection of Mycobacterium tuberculosis in coded sputum specimens. , 1994, The Indian journal of medical research.

[6]  D. Rienthong Rapid Detection for Early Appearance of Rifampin and Isoniazid Resistance in Mycobacterium Tuberculosis , 2009 .

[7]  R. Barker,et al.  A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR. , 1991, Molecular and cellular probes.

[8]  Yen-Hsu Chen,et al.  Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA-hybridization assay for molecular detection of Mycobacterium tuberculosis. , 2009, Journal of microbiological methods.

[9]  Bing Li,et al.  Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis. , 2009, Journal of microbiological methods.

[10]  Ehsan Aryan,et al.  A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex. , 2010, Microbiological research.

[11]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[12]  N. Arunrut,et al.  Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. , 2008, Journal of virological methods.

[13]  Yasuyoshi Mori,et al.  Sequence specific visual detection of LAMP reactions by addition of cationic polymers. , 2006, BMC biotechnology.