title: Detection of Mycobacterium tuberculosis by 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.
authors.affiliation: Innovative Learning Center, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand. thongchaika@swu.ac.th
authors.forename: Thongchai
authors.initials: T
authors.lastname: Kaewphinit
authors.forename: Narong
authors.initials: N
authors.lastname: Arunrut
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
authors.forename: Somchai
authors.initials: S
authors.lastname: Santiwatanakul
authors.forename: Pornpun
authors.initials: P
authors.lastname: Jaratsing
authors.forename: Kosum
authors.initials: K
authors.lastname: Chansiri
mesh_terms: D002845:Chromatography; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D013045:Species Specificity; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
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