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

    Research output: Contribution to journalArticle

    33 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)5608-5613
    Number of pages6
    JournalAnalytical Chemistry
    Volume79
    Issue number15
    DOIs
    Publication statusPublished - 2007 Aug 1

    Fingerprint

    DNA sequences
    Amplification
    Quenching
    Assays
    Bearings (structural)
    Fluorescence
    Humic Substances
    DNA
    Octoxynol
    Fluorescent Dyes
    Ammonia
    Oligonucleotides
    Urea
    Bacteria
    Genes
    Enzymes

    ASJC Scopus subject areas

    • Analytical Chemistry

    Cite this

    Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification. / Tani, Hidenori; Teramura, Tatsuya; Adachi, Ken; Tsuneda, Satoshi; Kurata, Shinya; Nakamura, Kazunori; Kanagawa, Takahiro; Noda, Naohiro.

    In: Analytical Chemistry, Vol. 79, No. 15, 01.08.2007, p. 5608-5613.

    Research output: Contribution to journalArticle

    Tani, Hidenori ; Teramura, Tatsuya ; Adachi, Ken ; Tsuneda, Satoshi ; Kurata, Shinya ; Nakamura, Kazunori ; Kanagawa, Takahiro ; Noda, Naohiro. / Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification. In: Analytical Chemistry. 2007 ; Vol. 79, No. 15. pp. 5608-5613.
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    AU - Kurata, Shinya

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