A novel method for the purification of DNA by capturing nucleic acid and magnesium complexes on non-woven fabric filters under alkaline conditions for the gene diagnosis of tuberculosis by loop-mediated isothermal amplification (LAMP).
A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg(2+)) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg(2+) complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg(2+) complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg(2+) was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.
Fukasawa
Tadashi
T
Asahi-Kasei Co., Ltd., Fuji 416-8501, Japan.
Oda
Naozumi
N
Wada
Yasunao
Y
Tamaru
Aki
A
Fukushima
Yukari
Y
Nakajima
Chie
C
Suzuki
Yasuhiko
Y
D001431:Bacteriological Techniques / Q000379:methods*; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D005374:Filtration / Q000379:methods*; D006801:Humans; D008274:Magnesium / Q000302:isolation & purification* / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D014376:Tuberculosis / Q000175:diagnosis*
D004247:DNA; D008274:Magnesium