Sumitomo Bakelite Company Limited

Yamaguchi University

Kenji Kinoshita

Kinoshita, Kenji

Kentaro Fujimoto

Kanehisa Yokoyama

Kazuhiko Fujiwara

Toru Yakabe

Shin Saito

Mutsunori Shirai

Hajime Fukunaga

A method of synthesizing a cDNA chain using an insoluble carrier having on the surface thereof a polymer substance containing a first unit having a group derived from a phosphoric ester composing the hydrophilic portion of phospholipid, and a second unit having a group derived from carboxylic acid having an electron-attractive substituent bound to a carbonyl group, which includes immobilizing a polynucleotide for DNA elongation; bringing a solution containing an RNA fragment, nucleotide monomers, and a reverse transcriptase or an enzyme having polymerase activity into contact with the surface of the insoluble carrier; and allowing the polynucleotide for DNA elongation immobilized on the surface of the carrier to elongate using the RNA fragment contained in the solution as a template, to thereby form a single-strand cDNA.

Method of synthesizing cDNA and method of synthesizing RNA chains, and nucleotide-immobilized carrier

[Object] It is to provide a method and a kit capable of detecting or quantifying a target RNA simply and rapidly from trace amounts of RNA in a sample, in a case such as when one or more kinds of pathogenic microorganisms are to be detected or quantified. [Solving Means] The method comprises the steps of 1) synthesizing cDNA from a sample containing the target RNA using a liquid-phase primer having a promoter sequence and a reverse transcriptase to obtain a cDNA-RNA complex, 2) degrading the RNA of the complex, 3) synthesizing a double-stranded DNA via the cDNA obtained in the step 2) and the solid-phase primer, 4) synthesizing RNA from the double-stranded DNA, 5) synthesizing cDNA via the RNA obtained in the step 4) and the solid-phase primer to obtain a cDNA-RNA complex, 6) degrading the RNA of the complex obtained in step 5), 7) synthesizing a double-stranded DNA via the cDNA obtained in the step 6) and the liquid-phase primer, and 8) quantifying the double-stranded DNAs obtained in the steps 3) and 7). These steps can be performed in a single reaction solution.

Method and kit for detection/quantification of target RNA

The present invention provides an RNA detection method detecting, from a reaction system containing a target sample, a target RNA chain originated from the target sample, using a surface having on the surface thereof a polymer substance which contains a first unit having a group derived from a phosphate ester composing the hydrophilic portion of a phospholipid and a second unit having a carboxylic acid derivative group composed of an electron-attractive substitutional group bound to a carbonyl group, while being provided with at least one reaction space, the reaction space having an immobilized nucleic acid primer immobilized therein.

RNA detection method

A method of detecting a gene including immobilizing a primer for DNA elongation onto an insoluble carrier having on the surface thereof a polymer substance containing a first unit having a phosphorylcholine group and a second unit having a carboxylic acid-derived group having an electron-attractive substituent bound to a carbonyl group; annealing the template DNA fragments or RNA fragments with the primer for DNA elongation, so as to elongate the DNA primer while incorporating therein an enzyme, thereby allowing coloration of a chromogenic reagent by its enzymatic action; and judging whether the DNA fragments or RNA fragments of the gene presents or not, based on the degree of coloration.

Growing community of inventors

Tokyo, Japan

Kentaro Fujimoto