One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.
In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.
Lee
Mei-Feng
MF
Department of Medical Laboratory Science and Biotechnology, College of Health Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chen
Yen-Hsu
YH
Hsu
Hui-Jine
HJ
Peng
Chien-Fang
CF
D000995:Antitubercular Agents / Q000494:pharmacology; D003124:Colorimetry / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004977:Ethambutol / Q000494:pharmacology; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013307:Streptomycin / Q000494:pharmacology; D014376:Tuberculosis / Q000382:microbiology
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D004977:Ethambutol; D004262:DNA Restriction Enzymes; D007538:Isoniazid; D013307:Streptomycin