Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates.
We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously. This method was used to detect Shiga toxin genes in Shiga toxigenic Escherichia coli isolates, demonstrating simultaneous detection of two different genes with rapidity and accuracy.
Kouguchi
Yoshihiro
Y
Fukuyama City Public Health Center, Fukuyama, Hiroshima, Japan. shiken-kensa@city.fukuyama.hiroshima.jp
Fujiwara
Takako
T
Teramoto
Miki
M
Kuramoto
Mika
M
D017931:DNA Primers / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007364:Intercalating Agents / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics*; D022641:Shiga Toxin 2 / Q000235:genetics*; D054323:Shiga-Toxigenic Escherichia coli / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification*; D013194:Staining and Labeling; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C024098:6-carboxyfluorescein; D004996:Ethidium