Toyo Boseki Kabushiki Kaisha

Motohiro Kondo

Toshiya Aono

Masaya Segawa

Yutaka Takarada

Koichi Yamanishi

Katsuya Daimon

Kazuhiro Kondo

Keiko Taya

The present invention provide a process for sequence-specific nucleic acid amplification capable of improving the detection sensitivity and increasing the signal. In particular, the present invention provides a reagent for nucleic acid amplification containing at least one member selected from the group consisting of EDTA, NTA, UDA, CyDTA, DTPA, GEDTA, TTHA and their salts, specifically a reagent for nucleic acid amplification comprising, in addition to the at least one compound, a forward primer having a DNA sequence homologous to a sequence of a target RNA; a reverse primer having a DNA sequence complementary to a sequence of the target RNA and having a promoter for RNA polymerase attached to its 5&prime; end; ribonucleotides; deoxyribonucleotides; a reverse transcriptase or RNA-directed DNA polymerase; a RNase H; a DNA polymerase or a reverse transcriptase having DNA-directed DNA polymerase activity; and a RNA polymerase. The present invention also provides a process for nucleic acid amplification characterized by carrying out a nucleic acid amplification reaction in the presence of at least one member selected from the group consisting of EDTA, NTA, UDA, CyDTA, DTPA, GEDTA, TTHA and their salts.

Reagent for nucleic acid amplification and process for nucleic acid amplification

Methods for nucleic acid amplification with thermostable ribonuclease H, in which single-stranded RNA (-) is prepared from RNA (+) as a target nucleic acid and the copy number of the single-stranded RNA (-) is increased through the use of thermostable ribonuclease H in combination with non-thermostable RNA-dependent DNA polymerase, non-thermostable DNA-dependent DNA polymerase and non-thermostable DNA-dependent RNA polymerase. In these methods, the number of amplification cycles is well increased and the sensitivity of detection can, therefore, be improved, as compared with the conventional methods. Also provided are methods for the detection of a target nucleic acid from RNA copies of a specific nucleic acid, obtained by any of the amplification methods, and reagent kits for use in these methods.

Methods for nucleic acid amplification with thermostable ribonuclease H

The present invention relates to two primers for amplifying a cytomegalovirus (CMV) nucleic acid suitable for a nucleic acid sequence-based amplification (NASBA) using a DNA-dependent RNA polymerase, a first primer containing a promoter sequence and a nucleic acid sequence consisting of at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1; a second primer containing a nucleic acid sequence consisting of at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, NO:3 or NO:4; a detecting probe and/or a capturing probe containing a nucleic acid sequence consisting of at least continuous fifteen bases selected from the nucleic acid sequence(s) of SEQ ID NO:5, NO:6 and/or NO:7 wherein said sequence is modified if necessary; a reagent kit for detecting a CMV containing the above-mentioned primers and probes; and a nucleic acid sequence-based amplification (NASBA) using said primers. The advantages of the present invention are that CMV can be detected in an easy, rapid and specific manner with a high sensitivity.

Oligonucleotides, methods and kits for amplifying and detecting a

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Otsu, Japan

Motohiro Kondo