Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification

Hidenori Tani, Tatsuya Teramura, Ken Adachi, Satoshi Tsuneda, Shinya Kurata, Kazunori Nakamura, Takahiro Kanagawa, Naohiro Noda

研究成果: Article

37 引用 (Scopus)

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We describe a novel technique for a simple, rapid, and reliable quantitative detection of specific DNA sequences using an alternately binding quenching probe (AB-QProbe) that binds to either the gene of interest (target) or an internal standard (competitor) in combination with loop-mediated isothermal amplification (LAMP). The AB-QProbe is a singly labeled oligonucleotide bearing a fluorescent dye at the 5′ end. The fluorescence intensity of the AB-QProbe reflects the ratio of the LAMP products from the target and competitor. We amplified the target and competitor by LAMP under isothermal conditions with high specificity, efficiency, and rapidity and calculated the starting quantity of the target from the fluorescence intensities at the beginning and end of LAMP. We call this technique alternately binding quenching probe competitive LAMP (ABC-LAMP). We quantified amoA, which encodes the ammonia-oxidizing enzyme in environmental bacteria, as a model target by ABC-LAMP, real-time PCR, and real-time turbidimetry of LAMP. By comparison, the accuracy of ABC-LAMP was found to be similar to that of real-time PCR. Moreover, ABC-LAMP enables the accurate quantification of DNA in the presence of DNA amplification inhibitors such as humic acid, urea, and Triton X-100 that compromise the values measured by real-time PCR and real-time turbidimetry of LAMP.

元の言語English
ページ(範囲)5608-5613
ページ数6
ジャーナルAnalytical chemistry
79
発行部数15
DOI
出版物ステータスPublished - 2007 8 1

ASJC Scopus subject areas

  • Analytical Chemistry

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