Patent FR-2902429-A1 (FR)
Title: Double pair of oligonucleotides to amplify two target sequences localized respectively in H5 gene and N1 genes of the genome influenza A virus, i.e. each the pair of oligonucleotides contains first and second oligonucleotides
IPC: G01N33/569
Filed: 2005-11-25
Abstract: Double pair of oligonucleotides to amplify two target sequences localized respectively in the H5 gene and N1 genes of the genome Influenza A virus, i.e. the pair of oligonucleotides amplifying H5 gene contains a first oligonucleotide and a second oligonucleotide; and the pair of oligonucleotides amplifying N1 gene contains a first oligonucleotide and a second oligonucleotide. Double pair of oligonucleotides to amplify two target sequences localized respectively in the H5 gene and N1 genes of the genome influenza A virus, i.e. the pair of oligonucleotides amplifying H5 gene contains a first oligonucleotide containing 10-50 nucleotides and comprising at least a consecutive nucleotide fragment comprising a fully defined 19 base pair (SEQ ID NO: 1) sequence as given in the specification, or its complementary sequence, and a second oligonucleotide containing 10-50 nucleotides and comprising at least a consecutive nucleotide fragment comprising a fully defined 18 base pair (SEQ ID NO: 2) sequence as given in the specification, or its complementary sequence; and the pair of oligonucleotides amplifying N1 gene contains a first oligonucleotide containing 10-50 nucleotides and comprising at least a consecutive nucleotide fragment comprising a fully defined 18 base pair (SEQ ID NO: 3) sequence as given in the specification or its complementary sequence and a second oligonucleotide containing 10-50 nucleotides and comprising at least a consecutive nucleotide fragment comprising a fully defined 21 base pair (SEQ ID NO: 4) sequence as given in the specification or its complementary sequence. Independent claims are included for: (1) oligonucleotide pair intended to be used as detection probes of two target sequences respectively localized in the H5 and N1 genes of the genome influenza A virus, i.e. the probe for detecting H5 gene comprising a fully defined 22 base pair (SEQ ID NO: 7) sequence as given in the specification, or its complementary sequence and the probe for detecting gene N1 comprises a fully defined 25 base pair (SEQ ID NO: 8) sequence as given in the specification, or its complementary sequence, each sequence comprising at least a marker; (2) detection of nucleic acids of the influenza A virus susceptible to be present in a sample, comprising subjecting the sample to an amplification reaction of nucleic acids using the pair of oligonucleotides, in the presence of the amplification reactive agents necessary for an amplification and detecting the presence of amplicons; (3) amplification method of two H5 and N1 genes of influenza A virus present in a sample, comprising: incubating the sample in an amplification buffer in the presence of two amplification initiators, each having 10-50 nucleotides, one comprising additionally a promoter sequence, the other of polarity opposed to the initiator associated with the promoter sequence, to hybrid itself respectively upstream and downstream of a zone of interest located in the H5 gene of the Influenza A virus and two amplification initiators, each having 10-50 nucleotides, one comprising additionally a promoter sequence, the other of polarity opposed to the initiator associated with the promoter sequence, to hybrid itself respectively upstream and downstream of a zone of interest located in the N1 gene of the Influenza A virus; adding the reactive agents such as an enzyme having a RNA dependent DNA polymerase activity, an enzyme having a DNA dependent DNA polymerase activity, an enzyme having a RNase H activity and an enzyme having a DNA dependent RNA polymerase activity; and maintaining the reaction mixture for amplification; and (4) detection kit of the H5 gene and N1 gene of the influenza A virus present in a sample, comprising: the double pair of oligonucleotides; two marked oligonucleotides or being able to be marked and having a nucleic acid sequence substantially complementary with at least a part of the amplified nucleic acid sequence; and the reactive agents for amplification.