Kit for detecting non-pathogenic or pathogenic influenza a subtype h5 virus

Abstract

Current methods for detecting influenza A subtype H5 virus, for example cell culture, haemagglutination-inhibition, fluorescent antibody and enzyme immunoassay, and reverse transcriptase polymerase chain reaction (RT-PCR) may have the disadvantages of low sensitivity and low specificity. Furthermore, such methods are relatively difficult to use, and may not be suitable for routine detection on a daily basis. The kit for detecting H5 virus of this invention may provide a user-friendly alternative that is relatively more sensitive and specific to H5 virus. The detection kit utilizes two specially designed primers A and B for the replication of H5 virus, and a specific capture probe for immobilizing the amplified viral RNA. An additional primer C is also designed for the detection of pathogenic H5 virus. The detection of H5 virus by the detection kit may be accomplished within one day if desired.

1 - 40 . (canceled) 
     
     
         41 . A purified and isolated DNA molecule or the complementary DNA molecule comprising essentially of any one of the DNA sequences set forth in SEQ ID NOS. 1 to 14. 
     
     
         42 - 55 . (canceled) 
     
     
         56 . A purified and isolated DNA molecule or the complementary DNA molecule including:
 (i) a first DNA sequence for binding to at least a portion of RNA sequence of haemagglutinin gene-containing influenza A subtype H5 virus; and   (ii) a second DNA sequence encoding a promoter DNA sequence of a RNA polymerase; such that the purified and isolated DNA molecule extends in the presence of an enzyme and DNA nucleotides to generate a DNA sequence including:
 (a) a DNA sequence complementary to at least a portion of the RNA sequence of H5 virus; and 
 (b) a DNA sequence encoding the promoter DNA sequence of a RNA polymerase when the first purified and isolated DNA molecule binds to at least a portion of the RNA sequence of H5 virus; 
   wherein the purified and isolated DNA molecule or the complementary DNA molecule consists of at least one of the DNA sequences set forth in SEQ ID NOs. 1 to 4 and 6 to 14.   
     
     
         57 . The molecule as claimed in  claim 56 , wherein the RNA polymerase is bacteriophage T7 RNA polymerase, and the second DNA sequence encodes the DNA sequence sets forth in SEQ ID NO.4. 
     
     
         58 . A purified and isolated DNA molecule or the complementary DNA molecule including:
 (i) a first DNA sequence encoding at least a portion of RNA sequence of haemagglutinin gene-containing influenza A subtype H5 virus; and   (ii) a second DNA sequence encoding a DNA sequence for binding to a detection molecule;   such that the purified and isolated DNA molecule extends in the presence of an enzyme and DNA nucleotides to generate a DNA sequence including:
 (a) a third DNA sequence encoding at least a portion of the RNA sequence of the H5 virus; and 
 (b) a fourth DNA sequence encoding the DNA sequence for binding a detection molecule when the purified and isolated DNA molecule binds to a DNA molecule including a DNA sequence complementary to at least a portion of the H5 virus; 
   wherein the first DNA sequence encodes at least one of the DNA sequences set forth in SEQ ID NOs. 5, 6, 7, 9, 10 and 11 which are bindable at regions between 914 to 940, 866 to 961, 846 to 981, 1017 to 1042, 970 to 1063 and 950 to 1083, of the haemagglutinin gene, respectively.   
     
     
         59 . The molecule as claimed in  claim 58 , wherein the second DNA sequence encodes the DNA sequence set forth in SEQ ID NO. 8. 
     
     
         60 . A purified and isolated DNA molecule according to  claim 41 , or the complementary DNA molecule thereof, wherein said DNA molecule encodes at least one of the DNA sequences set forth in SEQ ID NOs. 5, 6, 7, 9, 10 and 11 which are bindable at regions between 914 to 940, 866 to 961, 846 to 981, 1017 to 1042, 970 to 1063 and 950 to 1083, of the haemagglutinin gene, respectively. 
     
     
         61 . A purified and isolated DNA molecule or the complementary DNA molecule comprising:
 (i) a DNA sequence encoding at least a portion of RNA sequence of haemagglutinin gene-containing influenza A subtype H5 virus for binding to a target molecule, wherein the target molecule includes a nucleic acid sequence complementary to at least a portion of the H5 virus; and   (ii) an immobilizer;   such that the target molecule is immobilized when bound to the second purified and isolated DNA molecule; wherein the first DNA sequence encodes at least one of the DNA sequences set forth in SEQ ID NOs. 12, 13 and 14.   
     
     
         62 . A purified and isolated DNA molecule or the complementary DNA molecule thereof comprising:
 (i) a first DNA sequence encoding at least a portion of RNA sequence of haemagglutinin gene-containing influenza A subtype H5 virus for binding to a target molecule, wherein the target molecule includes a nucleic acid sequence complementary to at least a portion of the RNA sequence of influenza A subtype H5 virus; and   (ii) a signal generator;   such that a signal is generated from the target molecule when the target molecule is bound to the purified and isolated DNA molecule;   wherein said signal generator is selected from the group consisting of ruthenium-bipyridine complex [Ru(bpy) 3 ] 2+ , radioactive  32 P, chemiluminescent fluorescent, luciferin, luciferase, enzymatic alkaline phosphatase, enzymatic horseradish peroxidase and eletrodemiluminescence molecules.   
     
     
         63 . A purified and isolated DNA molecule according to  claim 41 , or the complementary DNA molecule thereof, wherein said DNA molecule is capable of binding at regions between 1107 to 1132, 1060 to 1140 and 1040 to 1160, 914 to 940, 866 to 961, 846 to 981, 1017 to 1042, 970 to 1063 and 950 to 1083 of haemagglutinin gene of influenza A subtype H5 virus, chosen from DNA sequences of SEQ ID NOs. 1 to 3, 5 to 7 and 9 to 11, respectively. 
     
     
         64 . (canceled) 
     
     
         65 . A purified and isolated DNA molecule that binds to a region of the haemagglutinin gene of influenza A subtype H5 virus, or a purified and isolated complement of said DNA molecule,
 wherein said isolated DNA molecule consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and   wherein said region of the haemagglutinin gene is selected from the group consisting of nucleotides 1107-1132, nucleotides 1060-1140, nucleotides 1040-1160, nucleotides 914-940, nucleotides 866-961, nucleotides 846-981, nucleotides 1017-1042, nucleotides 970-1063, and nucleotides 950-1083 of the haemagglutinin gene.   
     
     
         66 . (canceled) 
     
     
         67 . A kit for detecting influenza A subtype H5 virus in a biological sample, said kit comprising:
 (i) an isolating agent for isolating RNA molecules of the H5 virus from the biological sample and producing isolated RNA molecules therefrom;   (ii) a nucleic acid amplifying agent for amplifying the isolated RNA molecules of the H5 virus and producing amplified target molecules for detection, wherein the nucleic acid amplifying agent includes:
 (a) a first purified and isolated DNA molecule consisting of:
 (1) a first DNA sequence consisting of any one of the DNA sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; and 
 (2) a second DNA sequence encoding a promoter DNA sequence of an RNA polymerase; 
 and 
 
 (b) a second purified and isolated DNA molecule consisting of:
 (1) a third DNA sequence encoding at least a portion of the RNA sequence of the H5 virus; and 
 (2) a fourth DNA sequence encoding a fifth DNA sequence for binding to a detection molecule; 
 and 
 
   (iii) a nucleic acid detecting agent for detecting the target molecule; wherein the nucleic acid detecting agent includes the detection molecule.   
     
     
         68 . The kit for detecting H5 virus as claimed in  claim 67 , wherein the kit further comprises a lysis agent for stabilizing nucleic acids in the biological sample. 
     
     
         69 . The kit for detecting H5 virus as claimed in  claim 67 , further comprising an adsorbent for adsorbing nucleic acids. 
     
     
         70 . The kit for detecting H5 virus as claimed in  claim 67 , wherein the target molecule is a RNA molecule. 
     
     
         71 . The kit for detecting H5 virus as claimed in  claim 67 , wherein the RNA polymerase is bacteriophage T7 RNA polymerase. 
     
     
         72 . The kit for detecting H5 virus as claimed in  claim 67 , wherein the DNA sequence encoding the promoter DNA sequence of the RNA polymerase is set forth in SEQ ID NO. 4. 
     
     
         73 . The kit for detecting H5 virus as claimed in  claim 67 , further comprising a reverse transcriptase. 
     
     
         74 . The kit for detecting H5 virus as claimed in  claim 67 , further comprising a RNaseH. 
     
     
         75 . The kit for detecting H5 virus as claimed in  claim 67 , wherein the second purified and isolated DNA molecule or the fifth DNA sequence includes the DNA sequence set forth in SEQ ID NO. 8. 
     
     
         76 . The kit for detecting H5 virus as claimed in  claim 67 , further comprising a detection probe for generating a signal. 
     
     
         77 . The kit for detecting H5 virus as claimed in  claim 67 , further comprising an immobilizer whereby the target molecule can be immobilized when bound to the second purified and isolated DNA molecule.