title: Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes.
abstract: Because loop-mediated isothermal amplification (LAMP) can amplify substantial amounts of DNA under isothermal conditions, its applications for simple genetic testing have attracted considerable attention. A positive LAMP reaction is indicated by the turbidity caused by by-products or by the color change after adding a metallochromic indicator to the reaction solution, but these methods have certain limitations. Leuco crystal violet (LCV), a colorless dye obtained after sodium sulfite treatment of crystal violet (CV), was used as a new colorimetric method for detecting LAMP. LCV is reconverted into CV through contact with double-stranded DNA (dsDNA). Therefore, the positive reaction of LAMP is indicated by color change from colorless to violet. The assay is sensitive enough to detect LAMP products, with a detection limit of 7.1 ng/μl for dsDNA. It is also highly selective to dsDNA, and interference with single-stranded DNA and deoxynucleotide triphosphates (dNTPs) is not observed. LCV facilitates direct colorimetric detection of the main product rather than a by-product of the LAMP reaction; therefore, this method can be used under various reaction conditions such as those with added pyrophosphatase in solution. This colorimetric LAMP detection method using LCV is useful for point-of-care genetic testing given its simplicity.
authors.affiliation: Medical Device Development Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan. Electronic address: shigehiko_miyamoto@kn.kaneka.co.jp.
authors.forename: Shigehiko
authors.initials: S
authors.lastname: Miyamoto
authors.affiliation: Medical Device Development Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan.
authors.forename: Sotaro
authors.initials: S
authors.lastname: Sano
authors.affiliation: Medical Device Development Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Medical Device Development Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Jikihara
mesh_terms: D003124:Colorimetry / Q000379:methods*; D004396:Coloring Agents / Q000737:chemistry*; D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques; D011755:Pyrophosphatases / Q000378:metabolism; D014320:Triphenylmethyl Compounds / Q000737:chemistry*
chemical_list: D004396:Coloring Agents; D014320:Triphenylmethyl Compounds; C046945:triphenylmethane; D004247:DNA; D011755:Pyrophosphatases
keywords: Colorimetric detection; Genetic testing; LAMP; Triphenylmethane dye