Compositions and methods for detection and discrimination of influenza viruses

Abstract

The present disclosure relates to compositions and methods for detecting presence of an influenza virus in a sample, such as a biological sample obtained from a subject or an environmental sample. In some embodiments, the compositions and methods can be used to quickly identify particular subtypes of influenza virus (such as seasonal or variant influenza subtype H3, influenza subtype H5, Eurasian influenza subtype H7, North American influenza subtype H7, and/or influenza subtype H9) present in a sample. Probes and primers are provided herein that permit the rapid detection and/or discrimination of influenza virus subtype nucleic acids in a sample. Devices (such as arrays) and kits for detection and/or discrimination of influenza virus subtype nucleic acids are also disclosed herein.

We claim: 
     
         1 . A method for detecting an influenza virus nucleic acid in a sample, comprising:
 contacting the sample with:
 (a) an influenza subtype H7 probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 and at least one attached label, and a pair of primers comprising a forward primer comprising or consisting of SEQ ID NO: 14 and a reverse primer comprising or consisting of SEQ ID NO: 15; 
 (b) an influenza subtype H7 probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 106 and at least one attached label, and a pair of primers comprising a forward primer comprising or consisting of SEQ ID NO: 104 and a reverse primer comprising or consisting of SEQ ID NO: 105; and/or 
 (c) an influenza subtype H3 probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 3 and at least one attached label or comprising or consisting of the nucleic acid sequence of SEQ ID NO: 4 and at least one attached label and a pair of primers comprising a forward primer comprising or consisting of SEQ ID NO: 1 and a reverse primer comprising or consisting of SEQ ID NO: 2; 
   amplifying the influenza virus nucleic acid by polymerase chain reaction (PCR), real-time PCR, reverse transcriptase-PCR, real-time reverse transcriptase-PCR, ligase chain reaction, or transcription-mediated amplification; and   detecting hybridization between the amplified influenza virus nucleic acid and the one or more probes.   
     
     
         2 . The method of  claim 1 , further comprising discriminating between presence of an influenza virus Eurasian subtype H7 nucleic acid, an influenza virus North American subtype H7 nucleic acid, and/or an influenza subtype H3 nucleic acid. 
     
     
         3 . The method of  claim 1 , wherein the probe and pair of primers consists of:
 a probe consisting of the nucleic acid sequence of SEQ ID NO: 16 and at least one attached label, a forward primer consisting of SEQ ID NO: 14 and a reverse primer consisting of SEQ ID NO: 15, and hybridization of the probe to the influenza virus nucleic acid indicates the presence of Eurasian influenza subtype H7 in the sample;   a probe consisting of the nucleic acid sequence of SEQ ID NO: 106 and at least one attached label, a forward primer consisting of SEQ ID NO: 104, and a reverse primer consisting of SEQ ID NO: 105, and hybridization of the probe to the influenza virus nucleic acid indicates the presence of North American influenza subtype H7 in the sample;   a probe consisting of the nucleic acid sequence of SEQ ID NO: 3 and at least one attached label, a forward primer consisting of SEQ ID NO: 1, and a reverse primer consisting of SEQ ID NO: 2, and hybridization of the probe to the influenza virus nucleic acid indicates the presence of seasonal influenza virus subtype H3 in the sample; and/or   a probe consisting of the nucleic acid sequence of SEQ ID NO: 4 and at least one attached label, a forward primer consisting of SEQ ID NO: 1, and a reverse primer consisting of SEQ ID NO: 2, and hybridization of the probe to the influenza virus nucleic acid indicates the presence of variant influenza virus subtype H3 in the sample.   
     
     
         4 . The method of  claim 1 , further comprising contacting the sample with at least one probe comprising or consisting of the nucleic acid sequence of any one of SEQ ID NO: 9, 10, 13, 109, 29, 30, 31, 32, 33, 34, 35, 36, 94, 97, 100, or 103. 
     
     
         5 . The method of  claim 1 , further comprising contacting the sample with at least one primer comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 5-8, 11, 12, 107, 108, 37-52, 92, 93, 95, 96, 98, 99, 101, or 102. 
     
     
         6 . The method of  claim 1 , wherein the sample comprises a biological sample obtained from a subject or an environmental sample. 
     
     
         7 . The method of  claim 6 , wherein the biological sample obtained from the subject comprises bronchoalveolar lavage, tracheal aspirate, sputum, nasopharyngeal aspirate, oropharyngeal aspirate, or saliva. 
     
     
         8 . A probe for the detection of an influenza virus nucleic acid, wherein the probe is 20 to 40 nucleotides in length and comprises or consists of the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 106, SEQ ID NO: 3, or SEQ ID NO: 4, and at least one attached label. 
     
     
         9 . The probe of  claim 8 , wherein the at least one attached label is a radioactive isotope, enzyme substrate, co-factor, ligand, chemiluminescent agent, fluorophore, fluorescence quencher, hapten, enzyme, or combinations thereof. 
     
     
         10 . The probe of  claim 9 , wherein the at least one attached label is a fluorophore and/or a fluorescence quencher. 
     
     
         11 . A set of primers for the amplification of an influenza virus nucleic acid, comprising at least two primers comprising or consisting of the nucleic acid sequence of any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         12 . The set of primers of  claim 11 , wherein the set comprises:
 one or more forward primers comprising or consisting of the nucleic acid sequence of any one of SEQ ID NO: 14, SEQ ID NO: 104, or SEQ ID NO: 1; and   one or more reverse primers comprising or consisting of the nucleic acid sequence of any one of SEQ ID NO: 15, SEQ ID NO: 105, or SEQ ID NO: 2, wherein the set of primers is capable of hybridizing to and directing the amplification of the influenza virus nucleic acid.   
     
     
         13 . The set of primers of  claim 12 , wherein the forward and reverse primers are a pair of primers, and wherein the pair of primers:
 is specific for the amplification of influenza subtype Eurasian H7 and the set comprises a forward primer consisting of SEQ ID NO: 14 and a reverse primer consisting of SEQ ID NO: 15;   is specific for the amplification of influenza subtype North American H7 and the set comprises a forward primer consisting of SEQ ID NO: 104 and a reverse primer consisting of SEQ ID NO: 105; or   is specific for the amplification of influenza subtype H3 and the set comprises a forward primer consisting of SEQ ID NO: 1 and a reverse primer consisting of SEQ ID NO: 2.   
     
     
         16 . The set of primers of  claim 11 , further comprising at least one primer comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 5-8, 11, 12, 107, 108, 37-52, 92, 93, 95, 96, 98, 99, 101, or 102. 
     
     
         17 . A kit for detecting an influenza virus nucleic acid in a sample, comprising:
 at least one probe consisting of the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 106, SEQ ID NO: 3, or SEQ ID NO: 4 and at least one attached label; and   the set of primers of  claim 11 .   
     
     
         18 . The kit of  claim 17 , further comprising at least one probe comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 9, 10, 13, 109, 29, 30, 31, 32, 33, 34, 35, 94, 97, 100, or 103. 
     
     
         19 . The kit of  claim 17 , further comprising at least one primer comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 5, 6, 7, 8, 11, 12, 107, 108, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 92, 93, 95, 96, 98, 99, 101, or 102.