Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification.
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.
Tani
Hidenori
H
Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Teramura
Tatsuya
T
Adachi
Ken
K
Tsuneda
Satoshi
S
Kurata
Shinya
S
Nakamura
Kazunori
K
Kanagawa
Takahiro
T
Noda
Naohiro
N
D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D005456:Fluorescent Dyes / Q000737:chemistry; D006812:Humic Substances; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017830:Octoxynol; D010088:Oxidoreductases / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature; D014508:Urea
D015342:DNA Probes; D005456:Fluorescent Dyes; D006812:Humic Substances; D012329:RNA, Bacterial; D014508:Urea; D017830:Octoxynol; D004247:DNA; D010088:Oxidoreductases; C045582:ammonia monooxygenase