Sequences and methods for detecting influenza a and influenza b virus

Abstract

Nucleic acid amplification primers and methods for specific detection of influenza A and influenza B nucleic acid targets are disclosed. The primer-target binding sequences are useful for detection of influenza A and influenza B targets in a variety of amplification and hybridization reactions. The oligonucleotide sequences are able to differentiate between influenza A and influenza B strains through specific hybridization to one or the other virus strain, enabling specific detection of the presence of influenza A and/or influenza B in a specimen.

1 . An oligonucleotide selected from at least one of the following groups, the first group consisting of:
 a) an oligonucleotide consisting essentially of a nucleic acid sequence of any one of SEQ ID NOS: 19-24 or the complement thereof; and   b) an oligonucleotide having a nucleic acid sequence that specifically hybridizes to a nucleic acid sequence consisting essentially of SEQ ID NOS: 19-24, or to the complement thereof under conditions of 50 to 500 mM alkali metal ion at between 50° C. to 70° C.; and   the second group consisting of:   c) an oligonucleotide having a nucleic acid sequence selected from the group consisting of: SEQ ID NOS: 3-6 or the complement thereof; and   d) an oligonucleotide having a nucleic acid sequence that specifically hybridizes to any one of SEQ ID NOS: 3-6, or to the complement thereof, under conditions of 50 to 500 mM alkali metal ion at between 50° C. to 70° C.; and   the third group consisting of:   e) an oligonucleotide having a nucleic acid sequence selected from the group consisting of: SEQ ID NOS: 7-10 or the complement thereof; and   f) an oligonucleotide having a nucleic acid sequence that specifically hybridizes to any one of SEQ ID NOS: 7-10, or to the complement thereof, under conditions of 50 to 500 mM alkali metal ion at between 50° C. to 70° C.; and combinations thereof.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The oligonucleotide according to  claim 1 , wherein said oligonucleotide further comprises a detectable moiety wherein said detectable moiety is selected from the group consisting of: fluorescein isothiocyante (FITC)/tretramethykhodamine isothiocyanate (TRITC), FITC/Texas Red, FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), FITC/rhodamine X, FITC/tetramethylrhodamine (TAMRA), P-(dimethyl aminophenylazo) benzoic acid (DABCYL), 5-(2′-aminoetlryl) aminonapthalene, rhodamine, fluorescein,  32 P,  35 S, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, soybean peroxidase, luciferase, digoxigenin, biotin and 2,4-dinitrophenyl. 
     
     
         5 . (canceled) 
     
     
         6 . The oligonucleotide according to  claim 4 , wherein said oligonucleotide further comprises a restriction enzyme cleavage site wherein said restriction enzyme cleavage site is selected from the group of sites consisting essentially of: BsoBi, HincII, AvaI, NciI and Fnu4HI. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . A method for detecting the presence of influenza A or influenza B in a sample, said method comprising:
 a) amplifying a nucleic acid present in said sample using at least two nucleic acid primers, said nucleic acid primers consisting essentially of nucleic acid sequences selected from at least one of the groups consisting of: i) SEQ ID NOS: 19 and 20 ii) SEQ ID NOS: 21 and 22, iii) SEQ ID NOS: 23 and 20, and iv) SEQ ID NOS: 24 and 20; and b) detecting an amplified nucleic acid product, wherein detection of amplified product indicates presence of influenza A or influenza B in said sample.   
     
     
         14 . (canceled) 
     
     
         15 . The method according to  claim 13 , wherein detecting said amplified nucleic acid product is conducted by hybridizing said amplified nucleic acid product with at least one reporter probe comprising an oligonucleotide consisting essentially of a nucleic acid sequence of any one of SEQ ID NOS: 11, 12 or 19-24, and a reporter moiety. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . A method for specifically amplifying a target nucleic acid sequence of influenza A and/or influenza B, wherein said method comprises:
 a) hybridizing to said nucleic acid i) at least two amplification primers consisting essentially of a target binding sequence selected from at least one of each of the groups of sequences consisting of: SEQ ID NOS: 19 and 20, SEQ ID NOS: 23 and 20, and SEQ ID NOS: 24 and 20, and optionally, an additional sequence, and/or ii) at least two amplification primers consisting essentially of a target binding sequence selected from the group consisting of SEQ ID NOS: 21 and 22, and, optionally, an additional sequence, and;   b) extending the hybridized amplification primers on the target nucleic acid sequence to amplify said target nucleic acid sequence.   
     
     
         20 . The method according to  claim 19 , further comprising detecting the amplified target nucleic acid by hybridization of the amplified target nucleic acid sequence to at least one reporter probe oligonucleotide. 
     
     
         21 . The method according to  claim 20 , wherein the reporter probe oligonucleotide comprises a nucleic acid sequence according to SEQ ID NO: 15, optionally further comprising a detectable moiety. 
     
     
         22 . The method of  claim 21  wherein the detectable moiety is an additional sequence comprising a recognition site for a restriction endonuclease that is nickable by a restriction endonuclease. 
     
     
         23 . (canceled) 
     
     
         24 . The method according to  claim 19 , wherein the target nucleic acid is amplified by Polymerase Chain Reaction, Nucleic Acid Sequence Based Amplification, Transcription-Mediated Amplification, Rolling Circle Amplification, Strand Displacement Amplification or Ligation-Mediated Amplification. 
     
     
         25 . A kit comprising at least one oligonucleotide, wherein said oligonucleotide comprises a sequence selected from the group of sequences consisting of: SEQ ID NOS: 7-10 and 17-24. 
     
     
         26 . The kit according to  claim 25 , wherein said at least one oligonucleotide comprises oligonucleotides comprising sequences selected from one of the groups of sequences consisting of:
 a) SEQ ID NOS: 3, 4, 7, 8, 17 and 18, and   b) SEQ ID NOS: 5, 6, 9 and 10 and further comprising a reporter probe comprising a detectable moiety.   
     
     
         27 . (canceled) 
     
     
         28 . The kit according to  claim 25 , further comprising oligonucleotides that hybridize to a matrix protein gene of influenza A or influenza B. 
     
     
         29 . The kit according to  claim 25 , further comprising one or more oligonucleotides comprising nucleotide sequences selected from the group consisting of: SEQ ID NOS: 15 and 16. 
     
     
         30 . The kit according to  claim 26 , wherein said detectable moiety is selected from the group consisting of: fluorescein isothiocyante (FITC)/tretamethylrhodamine isothiocyanate (TRITC), FITC/Texas Red, FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isotbiocyanate (EITC), FITC/rhodamine X, FITC/tetramethylrhodamine (TAMRA), P-(dimethyl aminophenylazo) benzoic acid (DABCYL), 5-(2′-aminoethyl) aminonapthalene, rhodamine, fluorescein,  32 P,  35 S, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, soybean peroxidase, luciferase, digoxigenin, biotin and 2,4-dinitrophenyl. 
     
     
         31 . The kit according to  claim 25 , wherein said kit further comprises one or more oligonucleotides consisting essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 3-6. 
     
     
         32 . The kit according to  claim 25 , wherein said kit further comprises oligonucleotides consisting essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 11-16. 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . An oligonucleotide consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 3-24. 
     
     
         36 . An oligonucleotide comprising the sequence according to any one of SEQ ID NOS: 27-46, 63-68, 69 and 74-77. 
     
     
         37 . The oligonucleotide according to  claim 36 , wherein the oligonucleotide comprises one or more substitutions selected from the group consisting of:
 position 3 of SEQ ID NOS: 27-30 substituted with inosine or xanthine,   position 16 of SEQ ID NOS: 27-30 substituted with inosine or xanthine,   position 3 of SEQ ID NOS: 31-46 substituted with inosine or xanthine,   position 5 of SEQ ID NOS: 31-46 substituted, with inosine,   position 6 of SEQ ID NOS:31-46 substituted with inosine or xanthine,   position 15 of SEQ ID NOS: 31-46 substituted with inosine or xanthine,   position 13 of SEQ ID NOS: 47-62 substituted with inosine or xanthine,   position 16 of SEQ ID NOS:47-62 substituted with inosine,   position 17 of SEQ ID NOS: 47-62 substituted with inosine,   position 26 of SEQ ID NOS: 47-62 substituted with inosine,   position 14 of SEQ ID NOS: 63 and 64 substituted with inosine,   position 11 of SEQ ID NOS: 65-68 substituted with inosine,   position 14 of SEQ ID NOS:65-68 substituted with inosine or xanthine,   position 20 of SEQ ID NOS: 70-73 substituted with inosine or xanthine,   position 21 of SEQ ID NOS: 70-73 substituted with inosine,   position 1 of SEQ ID NOS: 74-77 substituted with inosine and   position 13 of SEQ ID NOS: 74-77 substituted with inosine or xanthine.   
     
     
         38 . At least one oligonucleotide selected from each of the group of oligonucleotides a), b) and c), comprising:
 a) an oligonucleotide comprising the sequence according to any one of SEQ ID NOS: 27-46,   b) an oligonucleotide comprising the sequence according to any one of SEQ ID NOS: 47-62, and   c) an oligonucleotide comprising the sequence according to any one of SEQ ID NOS: 63-68.   
     
     
         39 . The oligonucleotides according to  claim 38 , wherein the oligonucleotide comprises one or more substitutions selected from the group consisting of:
 position 3 of SEQ ID NOS: 27-30 substituted with inosine or xanthine,   position 16 of SEQ ID NOS: 27-30 substituted with inosine or xanthine,   position 3 of SEQ ID NOS: 31-46 substituted with inosine or xanthine,   position 5 of SEQ ID NOS: 31-46 substituted with inosine,   position 6 of SEQ ID NOS: 31-46 substituted with inosine or xanthine,   position 15 of SEQ ID NOS: 31-46 substituted with inosine or xanthine,   position 13 of SEQ ID NOS: 47-62 substituted with inosine or xanthine,   position 16 of SEQ ID NOS: 47-62 substituted with inosine,   position 17 of SEQ ID NOS: 47-62 substituted with inosine,   position 26 of SEQ ID NOS: 47-62 substituted with inosine,   position 14 of SEQ ID NOS: 63 and 64 substituted with inosine,   position 11 of SEQ ID NOS: 65-68 substituted with inosine and   position 14 of SEQ ID NOS: 65-68 substituted with inosine or xanthine.   
     
     
         40 . A collection of oligonucleotides comprising at least one oligonucleotide selected from each of the group of oligonucleotides a), b) and c), comprising:
 a) an oligonucleotide comprising the sequence according to SEQ ID NO: 69   b) an oligonucleotide comprising the sequence according to any one of SEQ ID NOS: 70-73, and   c) an oligonucleotide comprising the sequence according to any one of SEQ ID NOS: 74-77.   
     
     
         41 . The collection of oligonucleotides according to  claim 40 , wherein at least one oligonucleotide comprises one or more substitutions selected from the group consisting of:
 position 20 of SEQ ID NOS: 70-73 substituted with inosine or xanthine,   position 21 of SEQ ID NOS: 70-73 substituted with inosine,   position 1 of SEQ ID NOS: 74-77 substituted with inosine and   position 13 of SEQ ID NOS: 74-77 substituted with inosine or xanthine.   
     
     
         42 . A method for detecting the presence of influenza A in a sample, said method comprising:
 a) amplifying a nucleic acid present in said sample using at least two oligonucleotide primers, said oligonucleotide primers consisting essentially of nucleic acid sequences, at least one selected from each of the three groups consisting of:   i) SEQ ID NOS: 27-46, and   ii) SEQ ID NOS: 63-68; and   b) detecting an amplified nucleic acid product by hybridization to an oligonucleotide probe consisting essentially of a sequence selected from the group consisting of SEQ ID NOS: 47-62, wherein detection of amplified product indicates presence of influenza A in said sample.   
     
     
         43 . A method for detecting the presence of influenza B in a sample, said method comprising:
 a) amplifying a nucleic acid present in said sample using at least two oligonucleotide primers, said oligonucleotide primers consisting essentially of nucleic acid sequences, at least one selected from each of the groups consisting of:   i) SEQ ID NO: 69 and   ii) SEQ ID NOS: 74-77; and   b) detecting an amplified nucleic acid product by hybridization to an oligonucleotide probe consisting essentially of a sequence selected from the group consisting of SEQ ID NOS: 70-73, wherein detection of amplified product indicates presence of influenza B in said sample.