US 2010/0304377 A1Published application

Method of detecting norovirus rna

AssigneeTosoh Corporation

InventorsNoriyoshi Masuda, Kurando Une, Juichi Saito, Toshinori Hayashi

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Abstract

The amount of an RNA transcription product amplified in an RNA amplification process is measured using a nucleic acid probe labeled with an intercalating fluorescent dye. The RNA amplification process comprises the steps of using at least two sets of primer pairs comprising a first primer and a second primer (in which one of these primers carries a promoter sequence added to the 5′ end thereof), both of which have high hybridization efficiency to a nucleic acid sequence that is homologous to or complementary to each norovirus genotype RNA; forming a double-stranded DNA containing the promoter sequence with a reverse transcriptase; forming an RNA transcription product with an RNA polymerase by using the double-stranded DNA as a template; and forming the double-stranded DNA by successively using the RNA transcription product as a template in the DNA synthesis with the reverse transcriptase.

Claims5

Dependent claims

A method for detecting norovirus RNA in a sample, comprising the following steps:

(1) by use of a specified nucleic acid sequence derived from norovirus RNA as a template, producing a double-stranded DNA comprising a promoter sequence and said specified base sequence downstream from the promoter sequence, with a first primer and a second primer either one of which is added with the promoter sequence at the 5′ end, an RNA-dependent DNA polymerase, a ribonuclease H (RNase H) and a DNA-dependent DNA polymerase;
(2) producing an RNA transcript comprised of the specified base sequence with RNA polymerase using said double-stranded DNA as a template;
(3) amplifying the RNA transcript in a chain reaction using the RNA transcript as a template for the subsequent double-stranded DNA synthesis;
and
(4) measuring an amount of the RNA transcript, wherein the first primer consists of a mixture of at least two kinds of oligonucleotides selected from the group of oligonucleotides each of which is sufficiently homologous to any one of the sequences listed as SEQ ID Nos. 1 to 4, and the second primer consists of at least two kinds of oligonucleotides selected from the group of oligonucleotides each of which is sufficiently complementary to any one of the sequences listed as SEQ ID Nos. 5 to 9.

2 more elements

Dependencies are inferred from the claim text.