WO2008000023A1 - Detection of influenza virus - Google Patents

Detection of influenza virus Download PDF

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Publication number
WO2008000023A1
WO2008000023A1 PCT/AU2007/000882 AU2007000882W WO2008000023A1 WO 2008000023 A1 WO2008000023 A1 WO 2008000023A1 AU 2007000882 W AU2007000882 W AU 2007000882W WO 2008000023 A1 WO2008000023 A1 WO 2008000023A1
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sequence
pcr
template
seq
lane
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PCT/AU2007/000882
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French (fr)
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Ross Thomas Barnard
Lizeth Alejandra CASTILLO ALVAREZ
Richard You Jen LAI
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Biochip Innovations Pty Ltd
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Publication of WO2008000023A1 publication Critical patent/WO2008000023A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • This invention relates generally to a method for analyzing a target nucleic acid sequence. More particularly, the present invention relates to a method for determining the presence or absence of influenza virus type A, or nucleic acid sequences derived therefrom, in a subject. Methods of the invention facilitate the amplification of segments of the influenza type A haemagglutinin (HA), neuraminidase (NA) and PB2 genes from all variant type A subtypes using one set of oligonucleotides for each gene.
  • HA haemagglutinin
  • NA neuraminidase
  • PB2 PB2 genes from all variant type A subtypes using one set of oligonucleotides for each gene.
  • Methods of the invention further enable the determination of the pathogenicity and/or virulence status of those particular strains/subtypes of Influenza type A detected in accordance with the invention through analyzing the amplified segments which contain a specific target sequence(s).
  • the present invention further relates to kits for use in accordance with methods of the invention to amplify and analyze target nucleic acid sequences for the detection of influenza virus type A and all its subtypes.
  • Influenza infection is transmitted by tiny droplets of moisture spread from the respiratory tract of infected individuals by coughing, sneezing or even talking. When these are breathed in by a susceptible individual the viruses they contain can enter the cells of the respiratory tract and multiply. The individual will usually become ill within 2-3 days.
  • Two types of influenza viruses are of great public health concern; they are designated as influenza type A and influenza type B and can be readily distinguished from each other by laboratory tests but not by the clinical symptoms they produce.
  • Influenza type A viruses are contracted by in excess of 100 million people worldwide each year and such viruses have been demonstrated to mutate into the highly pathogenic subtypes. In birds, influenza A
  • H5N1 virus is a subtype of avian flu that is highly contagious and deadly. This viral strain does not usually infect humans, however, infections have been known to have occurred. The majority of cases relate to people in close contact with H5N1 infected poultry.
  • influenza viruses are covered with two types of glycoproteins, known as haemagglutinin (HA) and neuraminidase (NA), which, in electron microscope images, appear as a fringe or spikes projecting from the surface.
  • the HA protein is the most abundant and is responsible for attachment of the virus to the host cell membrane, whilst the NA protein is a host cell receptor destroying enzyme, thereby facilitating release of progeny virions.
  • Influenza type A is classified by its' subtypes. The subtypes are dependent on the sequences of the HA and NA surface proteins. Many different combinations of HA and NA proteins are possible, and each combination represents a different subtype. So far, 16 HA [H1-H16] and nine NA [N1-N9] subtypes have been detected in birds and mammals worldwide (Rohm et al, 1996, Virology, 217: 508-516).
  • HAO HA RNA is translated into a single precursor polypeptide, termed HAO, approximately 556 residues in length (Zambon, 1999, J. Antimicrob. Chemother, 44 (supp B): 3-9).
  • HAO must be further cleaved into two peptides, HAl and HA2, linked together by a disulphide bridge (Webster and Rott, 1987, Cell, 50(5): 665-666). The cleavage is carried out by the hosts' proteases.
  • the PB2 protein is a critical component of the influenza type A viral polymerase and is also associated with virulence. Approximately 75% of the highly virulent forms of H5N1 influenza virus isolates from Vietnam had a mutation consisting of a change to lysine at amino acid residue 627 in the PB2 protein. Mutations at amino acid residue 355 have been associated with the ability of the virus to replicate in humans.
  • Influenza type A is usually detected through the use of conventional diagnostic tools, cell culture and serologic testing which can take between two and fourteen days for results. In a disease outbreak situation, such testing procedures would not allow for the prevention of such a virulent infection from spreading throughout a population. Although, more efficient methods of virus detection have been discovered, a reliable and rapid method for the detection of all virus subtypes is still being investigated. [0007] At present, there are a range of methods by which the influenza virus can be analysed to determine virulence and pathogenicity.
  • Rapid assays have been designed for the detection of viral antigen in tissue impression smears and cryostat sections by use of immunofluorescence, or by antigen-capture enzyme-linked immunosorbent assays (ELISA) (Cattoli et al., 2004, Avain Pathol, 33(4): 432-437).
  • RNA molecules are becoming increasingly popular.
  • One such technique is based on the presence of influenza type A RNA in a sample, detected through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (Spackman et al., 2002, J. Clin. Microbiol, 40(9): 3256-3260).
  • RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
  • the amplified fragments of genes are analysed to characterise the specific subtypes.
  • the fragment can then be further analysed to establish the amino acid sequence at the endoproteolytic cleavage site, in turn indicating the pathology of the virus.
  • Molecular amplification techniques represent a rapid and reliable means for determining the presence of influenza virus with the added advantage that many samples can be analysed by less personnel on a large scale. Furthermore, molecular amplification techniques and sample preparation can also be readily automated.
  • a major problem faced in the design of oligonucleotide primers is the failure of the primers to successfully amplify regions within genes (for example the HA, NA and PB2 genes) of all subtypes of influenza type A. There are potentially 144 different combinations of HA and NA for any strain of influenza type A virus. The use of specific primers for each combination would neither be cost effective nor time effective.
  • the present invention is predicated in part on the unexpected discovery that diagnostically useful sequences in the HA, NA and PB2 genes of all influenza type A virus subtypes can be amplified through the use of a minimal set of specific oligonucleotide primers.
  • the invention may be employed without prior knowledge of the precise sequence of the gene(s) and/or the virus subtype.
  • the amplified sequences can be additionally analyzed to determine the pathogenicity of the virus, thereby providing an extremely rapid and sensitive method for influenza A diagnosis.
  • the present invention provides a method for detecting subtypes or strains of influenza type A virus, the method comprising: analyzing a segment of at least one gene selected from HA, NA and PB2 using a plurality of oligonucleotides, wherein at least one segment contains a target sequence that is predictive of pathogenicity, virulence or drug resistance.
  • the present invention provides a method of diagnosing influenza type A infection in an individual, the method comprising:
  • the present invention provides a test kit suitable for the detection of influenza type A from a biological sample, comprising: - oligonucleotides for amplifying a segment of at least one gene selected from
  • HA HA, NA and PB2, wherein an individual segment contains a target sequence that is predictive of pathogenicity, virulence or drug resistance;
  • the present invention provides a method for amplifying a target nucleic acid sequence in a test sample suspected of containing influenza type A virus. This method generally comprises:
  • DNA from a biological sample comprising of but not restricted to blood, bodily fluids, faeces, sputum, saliva and tissue biopsy;
  • an oligonuclotide set capable of annealing to at least one, two or three genes selected from HA, NA and PB2, wherein the individual segment(s) to be amplified contains a target sequence that is predictive of pathogenicity, virulence or drug resistance; 3. the appropriate concentration and quantity of reagents required for any suitable amplification method known in the art; subjecting the contents of the reaction vessel to a nucleic acid processing reaction to form one or more reaction products if the target nucleic acid is present in the test sample, wherein the reaction product(s) thus formed comprise the predictive target sequence; and
  • the method may include the purification of RNA from the sample and the subsequent reverse transcription of the RNA to cDNA. Accordingly, the method of amplification may be by reverse transcriptase PCR using the cDNA so produced as template.
  • the amplification reaction may incorporate one, two or even three gene products amplified in the same reaction vessel.
  • amplification reactions for the detection of HA, NA and PB2 gene segments may be carried out individually in separate reaction vessels, with specific amplification protocols.
  • the amplification techniques may include but are not restricted to PCR, Reverse Transcriptase PCR, Real-time PCR, Real-time Reverse Transcriptase PCR, strand displacement amplification, rolling circle amplification, nucleic acid bound amplification (NASBA), ligase chain reaction and QB replicase amplification.
  • Methods of the invention may be used to determine the pathogenicity and virulence status of the particular strain/subtype of Influenza type A through analyzing the genomic segments, which contain a specific target sequence.
  • Such techniques include but are not limited to the use of nucleotide probes, restriction endonuclease digest, Nested PCR and DNA sequencing.
  • the target sequence of the HA gene may encompass codons corresponding to amino acid residues at positions 226 and 228 of the HA protein, which are predictive for binding to receptors on avian cells or human cells.
  • the target sequence comprises the protease cleavage site within the HA gene responsible for cleaving the HAl and HA2 polypeptides.
  • the oligonucleotides specific for the HA gene are capable of detecting all known HA subtypes.
  • the oligonucleotides may comprise the sequences as set forth in any of SEQ ID NOs: 5 to 7 and 18 to 19.
  • the target sequence of the NA gene may encompass one or more codons corresponding to amino acid residues between positions 820 to 880 of the NA protein, in which mutations occur that determine resistance to Oseltamivir (Tamiflu).
  • the oligonucleotides specific for the NA gene are capable of detecting all known NA subtypes.
  • the oligonucleotides may comprise the sequences as set forth in any of SEQ ID NOs: 9 to 17 and 20 to 22.
  • the target sequence of the PB2 gene may encompass a codon corresponding to amino acid residue at position 627 and/or position 355 of the PB2 protein, which are predictive of replication efficiency of the virus, and associated with the ability of the virus to infect and replicate in humans, respectively.
  • the oligonucleotides may comprise the sequences as set forth in any of SEQ ED NOs: 1, 2, 3 and 4.
  • the present invention further relates to a kit that can be used in accordance with methods of the invention to amplify and analyze a target nucleic acid sequence for the detection of Influenza Virus Type A and all its subtypes.
  • the kit may contain the oligonucleotides of the invention and reagents for completing amplification techniques, preferably PCR.
  • the kit may also contain instructions on volumes of reagents and amplification methods.
  • the aspects and embodiments of the invention apply to any mammalian or avian species susceptible to infection by the influenza type A virus. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure l is a photographic representation of an agarose gel showing a 986 bp PB2 gene segment generated by one-step RT-PCR using forward primer 'PB2(1)F' (SEQ ID NO: 1) and reverse primer 'PB2(2)R' (SEQ ID NO: 2).
  • Figure 3 is a photographic representation of an agarose gel showing a 395bp PB2 gene segment generated by one-step RT-PCR using forward primer 'PB2(2)F' (SEQ ID NO: 4) and reverse primer 'PB2(2)R' (SEQ ID NO: 2).
  • Figure 4 is a photographic representation of an agarose gel showing a 395bp or 589 bp (approx.) products from PB2 gene segment generated by one-step RT-PCR using forward primer 'PB2(2)F' (SEQ ID NO: 4) and reverse primer 'PB2(2)R' (SEQ ID NO: 2) to generate the 395 bp product; or the PB2(1)F (SEQ ID NO: 1) and PB2(1)R (SEQ ID NO: 3) to generate the 589 bp product.
  • Lane 8 negative control, water in place of template; Lane 7, lO ⁇ l of PCR product using H7N7 viral RNA as template; Lane 6, lO ⁇ l of PCR product using H5N3 viral RNA as template; Lane 5, lO ⁇ l of PCR product using H7N4 viral RNA as template; Lane 4, lO ⁇ l of PCR product using H3N2 viral RNA as template; Lane 3, lO ⁇ l of PCR product using H7N3 viral RNA as template; Lane 2, lO ⁇ l of PCR product using H9N2 viral RNA as template; Lane 1, lO ⁇ l of PCR product using H5N1 viral RNA as template.
  • Lanes marked 'large M' and 'small M' are amplified from the M gene segment of influenza type A. All other lanes are amplified using the PB2 primer set. Lanes 1 to 8 are using primers PB2(2)F (SEQ ID NO: 4) and PB2(2)R (SEQ ID NO: 2). Lanes 9, 12, 13 and 14 are using primers PB2(1)F (SEQ ID NO: 1) and PB2(1)R (SEQ ID NO: 3).
  • Figure 5 is a photographic representation of an agarose gel showing a 986 bp PB2 gene segment generated by two-step RT-PCR using reverse primer 'PB2(2)R' (SEQ ID NO: 2) for cDNA synthesis; and using forward primer 'PB2(1)F' (SEQ ID NO: 1) and reverse primer 'PB2(2)R (SEQ ID NO: 2) to amplify the cDNA.
  • Lane M agarose gel marker, 100 bp Hyperladder II (Bioline Pty Ltd); Lane 1, 10 ⁇ L of PCR product using H1N9 viral RNA as template; Lane 2, lO ⁇ l of PCR product using H3N8 viral RNA as template; Lane 3, lO ⁇ l of PCR product using H5N3 viral RNA as template; Lane 4, 10 ⁇ l of PCR product using H6N5 viral RNA as template; Lane 5, lO ⁇ l of PCR product using H7N7 viral RNA as template; Lane 6, lO ⁇ l of PCR product using Hl 1N6 viral RNA as template; Lane 7, lO ⁇ l of PCR product using H12N9 viral RNA as template and; Lane 8, lO ⁇ l of PCR product using H15N9 viral RNA as template.
  • Figure 6 is a photographic representation of an agarose gel showing a 500 bp Haemagglutin gene segment generated by two-step RT-PCR using primer 'Unil2' (SEQ ID NO: 8) for cDNA synthesis; and forward primer 'RLHAP03F (SEQ ID NO: 5) and reverse primer 'RLHAP09R' (SEQ ID NO: 6) to amplify the cDNA.
  • Figure 7 is a photographic representation of an agarose gel showing a 500 bp Haemagglutinin gene segment generated by two-step RT-PCR using primer 'Unil2' (SEQ ID NO: 8) for cDNA synthesis; and forward primer 'RLHAP03F (SEQ ID NO: 5) and reverse primer 'RLHAP09R' (SEQ ID NO: 6) to amplify cDNA.
  • Agarose gel marker
  • Hyperladder II (Bioline Pty Ltd); Lane 1, Negative control, water in place of template DNA; Lane 2, Positive control, amplification of a NA gene segment generated by two-step RT-PCR using primer 'Unil2' (SEQ ID NO: 8) for cDNA synthesis; and forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR 1 (SEQ ID NO: 12) to amplify cDNA.
  • the primer combination 8F/10R gives a product of 219 bp; Lane 3, lO ⁇ l of HA gene segment PCR product using H5N3 viral cDNA as template; and Lane 4, lO ⁇ l of HA gene segment PCR product using H15N9 viral cDNA as template.
  • Figure 8 is a photographic representation of an agarose gel showing a 500bp Haemagglutinin gene segment generated by one-step RT-PCR using RLHAP03F (SEQ ID NO: 5) and reverse primer either RLHAP09MR (SEQ ID NO: 18) or RLHAP 1 OMR (SEQ ID NO: 19).
  • Lane M agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lO ⁇ l of HA gene segment RT-PCR product using H4N4 viral RNA as template and RLHAP03F/RLHAP09MR primers; Lane 2, lO ⁇ l of HA gene segment using H4N4 viral RNA as template and RLHAP03F/RLHAP10MR primers; Lane 3, 10 ⁇ l of negative control, water in place of template RNA and using RLHAP03F/RLHAP09MR primers. Lane 4, lO ⁇ l of negative control, water in place of template RNA and using RLHAP03F/RLHAP10MR primers.
  • Figure 9 is a photographic representation of an agarose gel showing a 219bp or 353 bp (approx.) products from Neuraminidase gene segment generated by two-step RT-PCR using primer 'NAl 3R' (SEQ LD NO: 16) for cDNA synthesis; and using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12) to generate the 219 bp product; or the 'NAlOF' (SEQ ID NO: 11) and 'NAl IR' (SEQ ID NO: 10) to generate the 353 bp product.
  • the gel illustrates the amplification of gene fragments from five different subtypes and seven different viruses.
  • Lane MM agarose gel marker, DMW-100L (GeneWorks); Lane 2, lO ⁇ l of PCR product using H1N9 viral RNA as template; Lane 3, lO ⁇ l of PCR product using H1N9 viral RNA as template; Lane 5, lO ⁇ l of PCR product using H3N8 viral RNA as template; Lane 6, 1 O ⁇ l of PCR product using H3N8 viral RNA as template; Lane 8, lO ⁇ l of PCR product using H5N3 viral RNA as template; Lane 9, lO ⁇ l of PCR product using H5N3 viral RNA as template, Lane 11, 1 O ⁇ l of PCR product using H6N5 viral RNA as template; Lane 12, lO ⁇ l of PCR product using H6N5 viral RNA as template; Lane 14, lO ⁇ l of PCR product using H7N7 viral RNA as template; Lane 15, 1 O ⁇ l of PCR product using H7N7 viral RNA as template; Lane 17, lO ⁇ l of PCR product using H12N9 viral
  • Figure 10 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12).
  • the gel illustrates the re- amplification of cDNA obtained from a gel purified PCR product from Figure 9.
  • Figure 11 is a photographic representation of an agarose gel showing a 353 bp Neuraminidase gene segment generated by PCR using forward primer 'NAlOF' (SEQ ID NO: 11) and reverse primer 'NAl IR' (SEQ ID NO: 10).
  • the gel illustrates the re- amplification of cDNA obtained from gel purified PCR product from Figure 9.
  • Figure 12 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer
  • the gel illustrates the amplification of gene fragments from three different putative subtypes of viruses (e.g., N3, N9, N7).
  • Lane MM agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lO ⁇ l of PCR product using H5N3 viral RNA as template; Lane 2, lO ⁇ l of PCR product using H15N9 viral RNA as template; Lane 3, lO ⁇ l of PCR product using H7N7 viral RNA as template; Lane 4, Negative control, water in place of template RNA.
  • Figure 13 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12).
  • the gel illustrates the amplification of gene fragments from three different putative subtypes of viruses (e.g., N4, N9 (previously unknown), Nl).
  • Figure 14 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12).
  • the gel illustrates the amplification of gene fragments from 3 different putative subtypes of viruses (e.g., N7, N4, Nl).
  • Lane MM agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lO ⁇ l of PCR product using H7N7 viral RNA as template; Lane 2, lO ⁇ l of PCR product using H7N4 viral RNA as template; Lane 3, lO ⁇ l of PCR product using H5N1C viral RNA as template and; Lane 4, Negative control, water in place of template RNA.
  • Figure 15 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12).
  • the gel illustrates the amplification of gene fragments from clinical samples.
  • Lane H 2 O Negative control, water in place of template RNA
  • Lane MM agarose gel marker, Hyperladder II (Bioline Pty Ltd)
  • Figure 16 is a photographic representation of an agarose gel showing a 253 bp (approximate) product from a Neuraminidase gene segment generated by PCR using forward primer 'NA8F-M13' (SEQ ID NO: 20) and reverse primer 'NA10R-M13' (SEQ ID NO: 21).
  • the gel illustrates the re-amplification of cDNA obtained from a gel purified PCR product of clinical samples.
  • Lane MM agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 21, lO ⁇ l of PCR product using Influenza A sample No. 21 (H3N?, unknown NA subtype) viral cDNA as template; Lane 30, lO ⁇ l of PCR product using Influenza A sample No.
  • Figure 17 is a schematic representation of a phylogenetic tree showing the position of the sequences obtained from several samples from Table 3.
  • amplification or “nucleic acid amplification” or “amplification reaction” refer to a biochemical reaction that produces many polynucleotide copies of a particular target nucleic acid sequence. If the target nucleic acid sequence is single-stranded complementary sequences may be produced in the reaction.
  • the reaction is a polymerase chain reaction (PCR) or a similar reaction that uses a polymerase to copy a nucleic acid sequence such as helicase dependent amplification (HDA), transcription mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA) and reverse transcription polymerase chain reaction (RT-PCR).
  • PCR polymerase chain reaction
  • HDA helicase dependent amplification
  • TMA transcription mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • RCA rolling circle amplification
  • RT-PCR reverse transcription polymerase chain reaction
  • a double stranded region formed through the hybridization of an oligonucleotide to a single-stranded form of the target nucleic acid sequence is required to prime (start) the reaction.
  • the terms "amplification” or “nucleic acid amplification” or “amplification reaction” refer to a biochemical reaction using a ligase or similar enzyme that covalently links two oligonucleotides or two oligonucleotide sub-sequences, such as a ligase chain reaction (LCR).
  • LCR ligase chain reaction
  • Ligase enzymes ligate the two oligonucleotides or oligonucleotide sub-sequences when they hybridize at adjacent sites in the target nucleic acid sequence.
  • the two oligonucleotides or oligonucleotide subsequences hybridize at sites that are one or more nucleic acid residues apart, i.e., they are not adjacent, then the single stranded region between the double stranded regions is converted to a double stranded region using a polymerase, and the ligase enzyme then links the adjacent oligonucleotides to form a continuous double stranded region.
  • the term "capturable sequence” refers to a nucleic acid sequence that is capable of hybridizing with another nucleic acid sequence.
  • capture oligonucleotide array means a plurality of capture oligonucleotides immobilized at discrete known locations on a solid surface, or on beads/colloids in suspension.
  • the capture oligonucleotides may be arranged in a two-dimensional spatially addressed array, e.g., a 2 x 2 array.
  • the capture oligonucleotides may be arranged in a tubular array in which a two-dimensional planar sheet is rolled into a three-dimensional tubular configuration.
  • the capture oligonucleotides is arranged on the inner or outer surface of a two- or three-dimensional reaction vessel of any convenient topology.
  • codon and “codons” refer to a sequence of three adjacent nucleotides constituting the genetic code that specifies the insertion of an amino acid in a specific structural position in a polypeptide chain during the synthesis of proteins.
  • complementary and complementarity refer to a sequence of nucleotides related by the base-pairing rules.
  • sequence “A-G-T-C” is complementary to the sequence “T-C-A-G”.
  • Complementarity may be "partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules.
  • nucleic acids there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • haemagglutinin is an antigenic glycoprotein found on the surface of the influenza virus and is responsible for binding the virus to the cell that is being infected.
  • haemagglutinin is given because the spikes are capable of adhering to red blood cells, causing them to be agglutinated.
  • Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA, DNA-RNA or a DNA-PNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • the base inosine (I) may also be used. Inosine can form base pairs with C or A or G or T (in descending order of stability).
  • the terms "match” and “mismatch” as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an "isolated oligonucleotide”, as used herein, refers to an oligonucleotide, which has been purified from the sequences that flank it in a naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
  • Influenza Type A genes include but are not limited to the hemagglutinins Hl, H2, and H3; the neuraminidase gene, which encodes for example, Nl and N2; the NP gene, which encodes the nucleoprotein; the M gene, which encodes the matrix proteins; the NS gene, which encodes two different non-structural proteins and one RNA molecule (PA, PBI and PB2) for each of the three subunits of the RNA polymerase.
  • the neuraminidase gene which encodes for example, Nl and N2
  • the NP gene which encodes the nucleoprotein
  • the M gene which encodes the matrix proteins
  • the NS gene which encodes two different non-structural proteins and one RNA molecule (PA, PBI and PB2) for each of the three subunits of the RNA polymerase.
  • Neuroaminidase is an antigenic glycoprotein enzyme found on the surface of the Influenza virus. The enzyme forms a mushroom-shaped projection on the surface of an influenza virus particle, and assists in the release of newly- formed virus particles from the surface of an infected cell.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • oligonucleotide typically refers to a nucleotide polymer in which the nucleotides and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule may vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotides.
  • Oligonucleotides can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences. Not all bases in the oligonucleotide need reflect the sequence of the template molecule to which the oligonucleotide will hybridize, the oligonucleotide need only contain sufficient complementary bases to enable hybridization to the template.
  • An oligonucleotide may also include mismatch bases at one or more positions, being bases that are not complementary to bases in the template, but rather are designed to incorporate changes into the DNA upon base extension or amplification.
  • An oligonucleotide may include additional bases, for example in the form of a restriction enzyme recognition sequence at the 5 ' end, to facilitate cloning of the amplified DNA.
  • oligonucleotide designate DNA, cDNA, RNA, mRNA, cRNA or PNA.
  • polynucleotide and nucleic acid typically refers to oligonucleotides greater than 30 nucleotides in length.
  • the term "pathogenicity” refers to the ability of the influenza A virus agent of known virulence to produce disease in a range of hosts under a range of environmental conditions.
  • the "PB2" gene encodes the PB2 protein which is a critical component of the viral polymerase of influenza.
  • primer an oligonucleotide which, when paired with a strand of DNA or RNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent.
  • the primer is typically single-stranded for maximum efficiency in amplification but may alternatively be double-stranded.
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers.
  • the oligonucleotide primer typically contains 15 to 35 or more nucleotides, although it may contain fewer nucleotides.
  • Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more.
  • Primers may be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis.
  • substantially complementary it is meant that the primer is sufficiently complementary to hybridize with a target nucleotide sequence.
  • the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential.
  • non-complementary nucleotides may be attached to the 5 1 end of the primer, with the remainder of the primer sequence being complementary to the template.
  • non-complementary nucleotides or a stretch of non-complementary nucleotides can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.
  • the terms "primer” and "oligonucleotide” may be used interchangeably.
  • references to describe sequence relationships between two or more nucleic acid sequences include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity” and “substantial identity”.
  • a “reference sequence” is at least 10 but frequently 15 to 20 and often at least 25 monomer units, i.e., nucleotides, in length.
  • two nucleic acid sequences may each comprise: (1) a sequence (i.e., only a portion of the complete nucleotide sequence) that is similar between the two polynucleotides and; (2) a sequence that is divergent between the two nucleic acid sequences, sequence comparisons between two (or more) nucleic acid sequences are typically performed by comparing sequences of the nucleic acid sequences over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 50 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • restriction endonuclease and “restriction enzyme” refer to a group of enzymes which cut DNA at a specific site called the recognition site. They are endonucleases in that they cut within a DNA molecule rather than cutting at the ends. There are three types of restriction endonucleases, and the majority of recognition sites at which they cut are palidromic.
  • sequence identity and “identity” are used interchangeably herein to refer to the extent that nucleic acid sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
  • sequence refers to the method of determining a DNA sequence through the process of establishing the nucleotide order of a given DNA fragment, called the DNA sequence.
  • DNA sequence Currently, almost all DNA sequencing is performed using the chain termination method, developed by Frederick Sanger. This technique uses sequence- specific termination of an in vitro DNA synthesis reaction using modified nucleotide substrates.
  • target sequence is used herein to refer to any nucleic acid or amino acid sequence of interest. It may be an entire gene, or portion thereof.
  • the target nucleic acid sequence may be a potion of a gene comprising a genetic mutation such as, but not limited to, nucleotide insertions, deletions and single nucleotide polymorphisms (SNPs).
  • the target nucleic acid sequence may also be a nucleic acid encoded by an entire gene, or portion thereof.
  • the target nucleic acid sequences contemplated by the present invention include, therefore, DNA, cDNA, RNA, mRNA and cRNA.
  • the target nucleic acid sequence may be mutated or contain an altered nucleic acid sequence.
  • strain refers to an influenza A virus strain classified as low pathogenic (LPAI) or highly pathogenic (HPAI) on the basis of specific molecular genetic and pathogenic criteria that requires specific testing as described by the present invention.
  • LPAI low pathogenic
  • HPAI highly pathogenic
  • stringency refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the degree of complementarity between hybridized nucleic acid sequences.
  • Stringent conditions refers to temperature and ionic conditions under which only polynucleotides and oligonucleotides that are substantially complementary or having a high proportion of complementary bases, preferably having exact complementarity, will hybridize and, in some embodiments, yield amplification products.
  • the stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used. Stringent conditions are well known to those of skill in the art.
  • Tm thermal melting point
  • the Tm is calculated by the nearest neighbour formulae as described by Breslauer et al., (1986, Proc. Nat. Acad. Sci.
  • RNA thermodynamic properties can be taken from Xia et al., (1998, Biochemistry, 37: 14719-14735). It will be understood that an oligonucleotide probe or primer will hybridize to a target sequence under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions.
  • Low stringency conditions for probe hybridization reactions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42 0 C, and at least about 1 M to at least about 2 M salt for washing at 42°C.
  • Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 rnM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 niM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • Medium stringency conditions for probe hybridization reactions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42°C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42°C.
  • Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65 0 C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 42°C.
  • BSA Bovine Serum Albumin
  • High stringency conditions for probe hybridization reactions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C.
  • High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65 0 C.
  • subtype and “subtypes” refer to groups of influenza type A viruses, wherein a subtype is classified on the basis of the identity of one or more proteins on the surface of the virus, typically the HA and NA proteins. Examples of such subtypes of influenza A viruses found in people are A(HlNl) and A(H3N2). Influenza B virus is not divided into subtypes.
  • viral disease refers to the ability of an infectious agent to do damage to the host. Viral virulence factors determine whether the infection occurs and how severe the resulting viral disease symptoms are. Virulence can be determined on a scale which ranges from high to low.
  • the present invention is directed to molecular methods and kits for determining the presence of influenza virus type A in a subject and to methods for amplifying target nucleic acid sequences derived from influenza virus type A.
  • Embodiments of the invention facilitate the analysis and amplification of segments of the influenza type A haemagglutinin (HA) genes, the neuraminidase (NA) genes and PB2 genes from all variant subtypes using at least one oligonucleotide primer for each gene.
  • embodiments of the invention may be employed without prior knowledge of the exact sequence of the gene(s) in question or of the strain/subtype of the virus present in the sample to be tested.
  • kits of the present invention may be used alone or in conjunction with other available testing procedures in identifying influenza type A viruses, viral particles and nucleic acid sequences derived there from.
  • Embodiments of the invention facilitate, for the first time, the detection and analysis of all known HA and NA subtypes of influenza type A virus.
  • analysis of segments of the influenza type A genes for the detection of influenza virus subtypes in accordance with the invention may be carried out by amplification, such as polymerase chain reaction.
  • amplification such as polymerase chain reaction.
  • analysis may be carried out using a variety of techniques for sequence analysis known in the art, with or without prior amplification, without departing from the scope of the present invention.
  • any source of nucleic acid in purified or non-purified form, can be utilized as the starting nucleic acid or acids, provided it contains or is suspected of containing the specific nucleic acid sequence desired.
  • the process may employ, for example, DNA or RNA, including mRNA, which DNA or RNA may be single stranded or double stranded.
  • a DNA-RNA hybrid which contains one strand of each may be utilized.
  • a mixture of any of these nucleic acids may also be employed, or the nucleic acid produced from a previous amplification reaction herein using the same or different primers may be so utilized.
  • the specific nucleic acid sequence to be amplified may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture.
  • the starting nucleic acid may contain more than one desired specific nucleic acid sequence which may be the same or different.
  • the nucleic acid(s) to be amplified may be obtained from any cell source or body fluid. Non- limiting examples of cell sources include blood cells, or any cells present in tissue obtained by biopsy.
  • Body fluids include, blood, saliva, urine, faeces, cerebrospinal fluid, semen and tissue exudates at the site of infection or inflammation.
  • Nucleic acids may be extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract the nucleic acids will depend on the nature of the source. Typically the nucleic acids to be analysed are isolated from the biological sample to be tested. However, it will be readily appreciated by those skilled in the art that methods of the invention may be carried out without prior nucleic acid isolation using techniques known in the art.
  • an RNA, DNA or cDNA sample is typically contacted with pairs of oligonucleotide primers, under conditions suitable for the amplification of the DNA or cDNA.
  • nucleic acid samples are amplified using polymerase chain reaction methods.
  • Oligonucleotides of the present invention hybridize to a target nucleic acid sequence when it is present in a test sample. Simultaneous extension of the annealed primers from a 3' terminus of each primer synthesizes an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the nucleic acid, if present, serves as a template for the synthesis of an extension product for the other primer of each pair; and analyzing the sample for the presence or absence of amplified products. The presence of amplified products indicates the presence of influenza A, or nucleic acids derived there from, in the sample.
  • Oligonucleotide primers may be provided in double-stranded or single- stranded form, although the single-stranded form is desirable.
  • oligonucleotide primers are labelled with radioactive species 32P, 14C, 35S, 3H, or other label), with a fluorophore (e.g., rhodamine, fluorescein) or with a chemillumiscent label (e.g., luciferase).
  • the optimal concentration for primers can be evaluated by performing single PCR reactions using each primer pair individually.
  • the concentration of each primer in the reaction mixture may range from about 0.01 pmol/ ⁇ l to about 100 pmol/ ⁇ l, or about 0.1 pmol/ ⁇ l to about 10 pmol/ ⁇ l, or about 0.2 pmol/ ⁇ l to about 7 pmol/ ⁇ l.
  • the primer pair should be in the orientation that permits amplification.
  • the forward primer should be in the 5'-3' orientation.
  • the reverse primer should be in an inverse complementary orientation.
  • One primer is designed to be complementary to the negative (-) strand and the other is complementary to the positive (+) strand.
  • the primers should be chosen as such that they are not complementary at the 3' end. A complementary region of equal to or greater than two nucleotides will cause an unwanted primer hybridization.
  • hybridization conditions utilizing at least two primer pairs of the invention include, for example, a hybridization temperature of about 50 0 C to about 65 0 C and a MgCl 2 concentration of about 1.5 mM to about 2.0 mM. Although lower temperatures and higher concentrations OfMgCl 2 can be employed, this may result in decreased primer specificity.
  • the oligonucleotides should be at least 10 nucleotides in length.
  • the oligonucleotides should amplify the desired region(s) of DNA or cDNA which contains the specified signature/target sequence. Annealing the primers to the denatured nucleic acid followed by extension with a suitable enzyme, and nucleotides results in newly synthesized + and - strands containing the target sequence.
  • Suitable enzymes are any enzymes for use in extension and amplification reactions as will be well known to those skilled in the art including, but are not limited to, the large fragment of DNA Polymerase I (Klenow), Taq polymerase, Pfu polymerase and Pfx polymerase.
  • the product of the chain reaction will be a discrete nucleic acid duplex with terminal corresponding to the ends of the specific primers employed.
  • Oligonucleotides of the invention include those suitable for the amplification of diagnostically useful sequences from the HA and NA genes and capable of detecting all known HA and NA subtypes of influenza type A virus.
  • Such oligonucleotides include those exemplified herein, comprising the sequences as set forth in SEQ ID NOs: 5 to 7, and SEQ ID NOs: 18 to 19 (HA subtype-specific) and SEQ ID NOs: 9 to 17 and 20 to 22 (NA subtype-specific).
  • primers suitable for amplification of diagnostically useful sequences of the PB2 gene as set forth in SEQ ID NOs: 1 to 4.
  • the present invention is not limited to the use of the specific oligonucleotides exemplified, but alternative oligonucleotide sequences may also be used, provided the oligonucleotides are designed appropriately so as to enable the amplification of target sequences from the HA, NA and/or PB2 genes.
  • the nucleotide sequence of a suitable oligonucleotide may share at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity with the sequence of an oligonucleotide as exemplified herein.
  • oligonucleotide of at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity.
  • the oligonucleotides of the invention for the amplification of segments of the influenza type A haemagglutinin (HA), neuraminidase (NA) and PB2 genes from type A subtypes can be chimerised with other sequences, for example sequencing primers (e.g., M13F and M13R) or tags for capture onto solid substrates.
  • sequencing primers e.g., M13F and M13R
  • Nucleic acids used in methods of the invention can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al, 1989, supra; and Ausubel et al, 1994, supra).
  • the nucleic acid is typically fractionated (e.g., poly A+ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA.
  • the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the influenza type A gene segments present in a given template sample.
  • nucleic acid amplification includes, but are not limited to, PCR (see, e.g., Saiki et al, 1985, Science, 230: 1350-1354; Mullis et al, 1987, Methods Enzymol, 155: 335-350), Strand Displacement Amplification (SDA and multiple SDA (MSDA); see, e.g., US Patent No. 5,422,252 and Little et al.,) Rolling Circle Amplification (RCA; see, e.g., Liu et al., 1996, J. Am. Chem. Soc, 118: 1587-1594 and US Patent No. 5,854,033 and US Patent No.
  • RNA sequence Based Amplification (NASBA; see, e.g., Sooknanan et al, 1994, Biotechniques, 17: 1077-1080), Ligase Chain Reaction (LCR; see, e.g., WO 89/09835) and Q ⁇ Replicase Amplification (see, e.g., Tyagi et al, 1996, supra).
  • PCR may be reverse transcriptase PCR (RT-PCR), Real-Time PCR or Real-Time RT-PCR.
  • RT-PCR reverse transcriptase PCR
  • Real-Time PCR Real-Time RT-PCR.
  • any useful combination of features of different amplification reactions may be used to increase the sensitivity and/or specificity of the method.
  • PCR polymerase chain reaction
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, (1989, supra). Alternative methods for reverse transcription utilise thermostable, RNA- dependent DNA polymerases.
  • the template-dependent amplification involves the quantification of transcripts in real-time.
  • RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al, Biotechnology, 10: 413-417).
  • Real-Time PCR technique Higuchi, 1992, et al, Biotechnology, 10: 413-417.
  • the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA.
  • LCR ligase chain reaction
  • Q ⁇ Replicase described in PCT Application No. PCT/US87/00880, may also be used.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5' ⁇ - thio-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al., (1992, Proc. Natl. Acad. Sci. U.S.A, 89: 392-396).
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • CPR a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • Still another amplification method described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, may be used.
  • modified primers are used in a PCR-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labelled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Proc. Natl. Acad. ScL U.S. A, 86:1173; Gingeras et al, PCT Application WO 88/10315).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989, Proc. Natl. Acad. ScL U.S. A, 86:1173; Gingeras et al, PCT Application WO 88/10315.
  • NASBA the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annea
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerisation.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • T7 or SP6 an isothermal cyclic reaction
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double- stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al., in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCR” (Frohman, M. A., In: “PCR Protocols: A Guide to Methods and Applications", Academic Press, N.Y., 1990; Ohara et al, 1989, Proc. Natl Acad. Sd. U.S.A, 86: 5673-567).
  • the polymerase dependent amplification comprises rolling circle amplification (RCA), in which hybridization of oligonucleotide primers to a circular nucleic acid molecule permits ligation, i.e., circularization, and a DNA polymerase, typically one that has strand displacement activity, to synthesise a first extension product using the circular nucleic acid molecule as a template.
  • the extension product is a long nucleic acid molecule containing multiple repeats of sequences complementary to the template circular nucleic acid molecule.
  • the first extension product can then serve as a template for the synthesis of further extension products, apropos of PCR, thereby permitting amplification of the original template circular nucleic acid molecule.
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used for amplifying target nucleic acid sequences. Wu et al., (1989, Genomics, 4: 560).
  • the influenza type A nucleic acid of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification. Next, the identified product is detected.
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, J Macromol. Sci. Pure. Appl. Chem, A31(l):1355-1376).
  • amplification products or "amplicons” are visualized in order to confirm amplification of the influenza type A gene sequences.
  • One typical visualisation method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products are integrally labelled with radio- or fiuorometrically-labelled nucleotides
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. In some embodiments, visualization is achieved indirectly.
  • a labelled nucleic acid probe is brought into contact with the amplified influenza type A sequence.
  • the probe is suitably conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antigen- binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule.
  • chromophore or radiolabel probes or primers identify the target during or following amplification.
  • the oligonucleotide sets of the present invention have been designed to amplify diagnostically useful sequences within the HA, NA and PB2 influenza type A genes.
  • the HA target region encompasses codons that correspond to amino acid residues which are predictive for binding to receptors on avian cells or human cells.
  • the target region also includes the HA cleavage site which determines the pathogenicity of influenza A viruses.
  • the NA target sequence encompasses the region in which occur mutations that determine resistance to Oseltamivir (Tamiflu).
  • the PB2 target sequence encompasses the region in which a mutation occurs in humans and birds consisting of an amino acid change to lysine or glutamic acid at position 627.
  • a labelled nucleic acid probe is brought into contact with the amplified influenza type A gene segment sequence.
  • the probe is suitably conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antigen- binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule.
  • chromophore or radiolabel probes or primers identify the target during or following amplification.
  • target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art.
  • Southern blotting involves the use of DNA as a target
  • Northern blotting involves the use of RNA as a target.
  • cDNA blotting is analogous, in many aspects, to blotting or RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilised on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter.
  • the blotted target is incubated with a probe (usually labelled) under conditions that promote denaturation and rehybridisation. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
  • a probe usually labelled
  • genotyping methods and allelic discrimination methods and technologies such as those described by Kristensen et al., (2001, Biotechniques, 30(2): 318-322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqMan®, liquid chromatography, and mass spectrometry.
  • biochip-based technologies such as those described by Hacia et al., (1996, Nature Genetics, 14: 441-447) and Shoemaker et al, (1996, Nature Genetics, 14: 450-456).
  • biochip technology By tagging amplified gene segments with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridisation. See also Pease et al, (1994, Proc. Natl. Acad. ScL U.S.A, 91: 5022-5026); Fodor et al., (1991, Science, 251: 767- 773). Briefly, nucleic acid probes to influenza type A polynucleotides are made and attached to biochips.
  • the nucleic acid probes attached to the biochip are designed to be substantially complementary to specific influenza type A gene segments amplified or the target sequence contained therein or to other probe sequences, (for example in sandwich assays), such that hybridisation of the target sequence and the probes of the present invention occurs.
  • This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridisation between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridisation can occur under even the least stringent of hybridisation conditions, the sequence is not a complementary target sequence.
  • more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target.
  • the probes can be overlapping (i.e., have some sequence in common), or separate.
  • nucleic acids can be attached to or immobilised on a solid support in a wide variety of ways.
  • immobilised and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below.
  • the binding can be covalent or non- covalent.
  • non-covalent binding and grammatical equivalents herein is meant one or more of either, electrostatic, hydrophilic, and hydrophobic interactions.
  • non- covalent binding includes the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
  • covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds.
  • Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilisation may also involve a combination of covalent and non-covalent interactions.
  • the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
  • the nucleic acids can either be synthesised first, with subsequent attachment to the biochip, or can be directly synthesised on the biochip.
  • the biochip comprises a suitable solid or semi-solid substrate or solid support.
  • substrate or “solid support” is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
  • the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalised glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc.
  • the substrates allow optical detection and do not appreciably fluoresce.
  • the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well.
  • the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimise sample volume.
  • the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
  • oligonucleotide probes are synthesised on the substrate, as is known in the art.
  • photoactivation techniques utilising photopolymerisation compounds and techniques can be used.
  • the nucleic acids are synthesised in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505 and U.S. Pat. NOs. 5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis of the Affymetrix GeneChip technology.
  • biochip analysis oligonucleotide probes on the biochip are exposed to or contacted with amplified segments of one or more influenza type A genes under conditions favouring specific hybridisation.
  • cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases.
  • cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilised oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridisation conditions.
  • fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.
  • the target Influenza type A polynucleotides can be detectably labelled so that their hybridisation to individual probes can be determined.
  • the target polynucleotides are typically detectably labelled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu34), a radioisotope and a direct visual label.
  • a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu34), a radioisotope and a direct visual label.
  • a direct visual label use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide, hi some embodiments in which an enzyme is used as a direct visual label, biotinylated bases are incorporated into a target polynucleotide. Hybridisation is detected by incubation with streptavidin-reporter molecules.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by Dower et al., (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al.,) 5,326,692 (Brinkley et al.,). Alternatively, reference may be made to the fluorochromes described in U.S. Patent NOs.
  • fluorescent labels include, for example, fluorescein phosphoramidites such as FluoreprimeTM (Pharmacia), Fluoredite (Millipore) and FAM (Applied Biosystems International) [0112]
  • Radioactive reporter molecules include, for example, 32P, which can be detected by an X-ray or phosphorimager techniques.
  • the hybrid- forming step can be performed under suitable conditions for hybridising oligonucleotide probes to test nucleic acid including DNA or RNA.
  • suitable conditions for hybridising oligonucleotide probes to test nucleic acid including DNA or RNA.
  • high discrimination hybridisation conditions are used.
  • Wallace et al. (1979, Nucl. Acids Res. 6: 3543) who describe conditions that differentiate the hybridisation of 11 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch.
  • Wood et al. (1985, Proc. Natl. Acid. Sci.
  • a hybridisation reaction can be performed in the presence of a hybridisation buffer that optionally includes a hybridisation optimising agent, such as an isostabilising agent, a denaturing agent and/or a renaturation accelerant.
  • a hybridisation optimising agent such as an isostabilising agent, a denaturing agent and/or a renaturation accelerant.
  • isostabilising agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts.
  • Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules.
  • Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulphoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts.
  • Hybridisation accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol.
  • CAB cetyltrimethylammonium bromide
  • DTAB dodecyl trimethylammonium bromide
  • polylysine polylysine
  • spermine spermine
  • spermidine single stranded binding protein
  • SSB single
  • Hybridisation buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM.
  • a hybridisation mixture containing the target influenza type A marker polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridisation between the target sequences in the amplified polynucleotide segments and any complementary probes. Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound.
  • incubation will be at temperatures normally used for hybridisation of nucleic acids, for example, between about 20 0 C and about 75 0 C, example, about 25 0 C, about 30 0 C, about 35 0 C, about 40 0 C, about 45 0 C, about 50 0 C, about 55 0 C, about 60 0 C, or about 65 0 C.
  • 20 0 C to 50 0 C is desirable.
  • lower temperatures are preferred.
  • a sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridisation between the target sequences in the target polynucleotides and any complementary probes.
  • the hybridisation may be carried out at about 45 0 C +/- 10 0 C in formamide for 1-2 days.
  • the probes are washed to remove any unbound nucleic acid with a hybridisation buffer, which can typically comprise a hybridisation optimising agent in the same range of concentrations as for the hybridisation step. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridised to a target polynucleotide.
  • a hybridisation buffer which can typically comprise a hybridisation optimising agent in the same range of concentrations as for the hybridisation step. This washing step leaves only bound target polynucleotides.
  • the probes are then examined to identify which probes have hybridised to a target polynucleotide.
  • a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a coloured colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography.
  • a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light.
  • a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe: target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer.
  • electronic detection of the signal may not be necessary.
  • the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile.
  • oligonucleotide probes specific for different influenza type A gene sequences are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a
  • a detection system that can be used by a 'chip reader' is described for example by Pirrung et al., (U.S. Patent No. 5,143,854).
  • the chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal.
  • Exemplary chip readers are described for example by Fodor et al., (U.S. Patent No., 5,925,525).
  • the reaction may be detected using flow cytometry.
  • target sequences of the invention may be determined through the use of restriction endonucleases.
  • Restriction endonucleases used under specific conditions which are well known in the art may be efficient in digesting the target DNA sequences of the highly pathogenic strains of influenza as opposed to the low pathogenic strains, or vice versa.
  • the target sequences of the invention may also be determined through the use of nested PCR.
  • Nested PCR is the method by which two pairs of PCR primers are used for the amplification of a single locus. The first pair amplify the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first PCR product and produce a second PCR product that will be shorter than the first one.
  • the logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.
  • kits of the invention may optionally include appropriate components including but not restricted to, positive and negative controls, dilution buffers and the like. Also included may be components suitable for subjecting nucleic acids to a nucleic acid processing and/or amplifying reaction. These components include various polymerases such as, but not limited to, Taq polymerase, reverse transcriptase, DNA ligase etc. (depending on the nucleic acid processing reaction technique employed), nucleotide precursors, salts and buffer solutions.
  • polymerases such as, but not limited to, Taq polymerase, reverse transcriptase, DNA ligase etc. (depending on the nucleic acid processing reaction technique employed), nucleotide precursors, salts and buffer solutions.
  • Kits may further include reagents for carrying out analysis of the amplified nucleic acids, such as an appropriate restriction enzyme(s), reaction buffer(s) for restriction enzyme digestion, and reagents for use in separating digested fragments (e.g., agarose) and/or nucleic acid sequencing.
  • Kits of the invention may also comprise distinct containers for the individual components, hi some embodiments, the kits comprise instructions for performing the methods of the present invention.
  • the kit of the present invention comprises a reaction vessel having HA, NA and PB2 oligonucleotides immobilized thereto and containing a pre-prepared mixture of reagents, suitably in lyophilized form.
  • RT-PCR primers were designed to amplify a part of the PB2 segment from a wide range of subtypes of influenza A virus.
  • the PB2 influenza forward primer PB2(1)F comprises the sequence 5'- AG[Y]TC[I]TC[Y]TT[Y]AG[Y]TT[Y]GG - 3' (SEQ ID NO: 1).
  • the PB2 influenza reverse primer PB2(2)R comprises the sequence 5'- AGTAT[Y]CTCAT[Y]CC[W]GANCC -3' (SEQ ID NO: 2).
  • the expected size of the product generated from these primers is approximately 986bp (refer to Figure 1). However, this may vary depending upon the viral isolate present in a sample of interest as the length of the PB2 coding sequence may vary slightly between isolates.
  • PB2(1)R comprises the sequence 5'- C[I]GG[I]GA[Y]A[R][K]AG[Y]A[Y] [R]TT[Y]C -3' (SEQ ID NO: 3).
  • the expected size of the product generated from the above primer pair is approximately 589 bp (refer to Figure 2). However, this may vary depending upon the viral isolate present in a sample.
  • PB2(2)F which comprises the sequence 5'- GA[I]GT[I]AG[Y]GA[R]AC[M]CA[R]GG -3' (SEQ ID NO: 4).
  • the expected size of the product generated from the primer pair PB2(2)F and PB2(2)R is approximately 395bp (refer to Figure 3). However, this may also vary depending upon the viral isolate present in a sample.
  • Viral RNA was extracted from the sample (e.g., amniotic fluid or clinical sample) using a QIAGEN RNA easy extraction kit by following the manufacturer's instructions. 100 ⁇ l of sample was inactivated by addition of 600 ⁇ l of a guanidium denaturant and 6 ⁇ l of 2-mercaptoethanol prior to use in the QIAGEN extraction protocol. The extracted RNA was resuspended in 50 ⁇ l of RNase free water (QIAGEN) and 2 ⁇ l of the resuspended RNA was used in the RT-PCR reaction.
  • QIAGEN RNase free water
  • Sense primer selected PB2 primer 2 ⁇ l 200 pmoles
  • Anti-sense primer selected PB2 primer 2 ⁇ l 200 pmoles
  • Platinum Taq Polymerase Mix (Invitrogen) 0.2 ⁇ l (5 U/ ⁇ l) ⁇ 5OmM MgCl 2 (2 ⁇ l)
  • influenza PB2 forward primers and the influenza PB2 reverse primers are incorporated into a double stranded PCR product.
  • the final PCR reaction sample is visualised by UV transillumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 2OmM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes.
  • TAE IX tris-acetate
  • DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100 L, GeneWorks) that is used as a base pair size reference.
  • the reaction is alternatively set up as a one-step procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps.
  • the thermal cycler was programmed so that cDNA synthesis is followed immediately by PCR amplification, as follows:
  • N may be substituted for "I" in these above primer sequences and the primers generate an approximate 589 bp product (refer to Figure 2).
  • N may be substituted for "I” in this above primer sequence.
  • PB2(2)-R 5 '-AGTATYCTCATYCCWGANCC-S ' SEQ ID NO: 2
  • the reaction is set up as a 'one-step' procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps. Note the different temperature for extension (as this is carried out at the temperature recommended for the superscript enzyme rather than the Taq used in the two step procedure (below)).
  • RT-PCR primers are designed to amplify a part of the HA segment from a wide range of subtypes of influenza A virus.
  • the HA influenza forward primer RLHAP03F comprises the sequence 5'- T[I]TGGGG[I][R]T[I][M]A[Y]CA[Y][Y]C-3' (SEQ ID NO: 5).
  • the HA influenza reverse primer RLHAP09R comprises the sequence 5'- CCA[I]CCA[I]CC[I][Y][Y][Y]TC -3' (SEQ ID NO: 6).
  • the expected size of the product generated from these primers is approximately 500 bp. However, this may vary depending upon which virus is present in a sample of interest as the length of the HA coding sequence may vary slightly between isolates.
  • RLHAP09R comprises the sequence 5'- CCI[K][I]CCA [I]CC[I] [Y][Y][Y]TC -3' (SEQ ID NO: 7).
  • two reverse primers have been designed, which comprise the sequence of 5'-CC[I][K][I]CCA[I]CC[I][Y][Y][Y]TC[I]AT-3 '(SEQ ID NO: 18) and 5'-A[I][I]CC[I][K][I]CCA[I]CC[I][B][Y][Y]TC-3' (SEQ ID NO: 19).
  • Viral RNA was extracted from the sample (e.g., amniotic fluid or clinical sample) using a QIAGEN RNA Easy extraction kit by following the manufacturer's instructions. 100 ⁇ l of sample was inactivated by addition of 600 ⁇ l of a guanidium denaturant and 6 ⁇ l of 2-mercaptoethanol prior to use in the QIAGEN extraction protocol. The extracted RNA was resuspended in 50 ⁇ l of RNAse free water (QIAGEN). 2 ⁇ l of the resuspended RNA was used in the RT-PCR reaction.
  • QIAGEN RNAse free water
  • the Unil2 primer sequence is 5'-AGCAAAAGCAGG-S' (SEQ ID NO: 8). Denaturation:
  • Anti-sense primer RLHAP09R 0.75 ⁇ l (75 pmoles) Platinum Taq Polymerase Mix (Invitrogen) 0.5 ⁇ l (5 U/ ⁇ l)
  • the final PCR reaction sample is visualised by UV transillumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 20 mM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes.
  • DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100L, Gene Works) that is used as a base pair size reference.
  • TAE IX tris-acetate
  • the reaction is alternatively set up as a one-step procedure without the separate production of cDNA. This procedure is faster; requiring shorter cycle times and no manipulation between the RT and PCR steps (refer to Figure 8).
  • RT-PCR primers were designed to amplify a segment of the NA gene from a wide range of subtypes of influenza A virus.
  • Two pairs of primers were designed for the amplification of the NA gene segment, these consist of:
  • the NA influenza forward primer 'NA8F' comprises the sequence 5'- G[R]AC[H]CA[R]GA[R]TC[I][K][M][R]TG -3' (SEQ ID NO: 9) and the NA influenza reverse primer 'NAlOR' comprises the sequence 5'- CC[I][I][K]CCA[R]TT[R]TCfY]CT[R]CA -3' (SEQ ID NO: 12).
  • the NA influenza forward primer 'NAlOF' comprises the sequence 5'-
  • NA influenza reverse primer 'NAUR' comprises the sequence 5'- CC[D]A[S]A[R]TA[l]CC[l]GACCA[R]T-3' (SEQ ID NO: 10).
  • the expected size of the product generated from the 'NA8FVNA10R' primers is approximately 219 bp.
  • the expected size of the product generated from the 'NA8FVNA10R' primers is approximately 219 bp.
  • 'NAlOFVNAl IR' primers is approximately 353 bp. However, this may vary depending upon the viral isolate present in a sample of interest as the length of the NA coding sequence may vary slightly between isolates. Extraction of RNA from sample:
  • Viral RNA was extracted from the sample (e.g., amniotic fluid or clinical sample) using a QIAGEN RNA easy extraction kit (for the samples derived from amniotic fluid) or a Roche MagNA Pure LC total nucleic acid isolation kit (for clinical samples) by following the manufacturer's instructions.
  • sample e.g., amniotic fluid or clinical sample
  • QIAGEN RNA easy extraction kit for the samples derived from amniotic fluid
  • Roche MagNA Pure LC total nucleic acid isolation kit for clinical samples
  • RNA was extracted from nasopharyngeal aspirates (NPA) specimens provided by the Molecular Diagnostic Unit of Queensland Health Pathology and Scientific Services (QHPSS) as blind specimens.
  • NPA nasopharyngeal aspirates
  • QHPSS Molecular Diagnostic Unit of Queensland Health Pathology and Scientific Services
  • the clinical specimens were collected from suspect cases of viral respiratory disease during a period of September to October 2006, mainly from Queensland (Australia) population.
  • the initial NA subtyping described in Figures legends: 15 and 16 was done by QHPSS either by NA inhibition test or NA serum antibody. There are some NA subtypes unknown by these methods.
  • the RNA was extracted from 200 ⁇ l of NPA samples using Roche MagNA Pure LC total nucleic acid isolation kit and RNA was eluted in 100 ⁇ l of elution buffer. 1 ⁇ l of the resuspended RNA was diluted in 1 ⁇ l of ultra pure water, and then the 2 ⁇ l total volume were used in the one- step
  • cDNA synthesis 1. Reverse Transcription mix lOO ⁇ M 'NAl 3R' gene specific primer 2 ⁇ l
  • the PCR experiments included negative controls containing all the components of the reaction mixture except template DNA, and primers, since different pairs of primers were tested at the same time.
  • the volume of the components omitted was replaced by an equivalent volume of ultra pure distilled water DNAse and RNAse free.
  • the final PCR reaction samples are visualised by UV transilumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 2OmM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes.
  • DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100L, Gene Works) that is used as a base pair size reference.
  • oligonucleotide sequences for the amplification of target sequences within the NA gene have also been designed as follows: 'NA5F': 5'-CA[Y][D][S][I]AATGR[I]AC[M][R]T[I][M]A[I]GA-3' (SEQ ID NO: 13) 'NAI lF': 5'-A[Y]TGGTC[I]GG[I]TA[Y]T[S]T[H]GG-3' (SEQ ID NO: 14) 'NA8R': 5'-CA[Y][K][M][I]GA[Y]TC[Y]TG[D]GT[Y]C-3' (SEQ ID NO: 15) •NA13R': 5'-[K]G[I][W][M][I]T[K][S]C[M][I]GATGG[I][K]C-S 1 (SEQ ID NO: 16)
  • NA5F, NAl IF, NA8R, NA13R and NA9R may be used in a variety of combinations, including NA8F and NAlOR, NA5F and NA8R, NA8F and NA9R and NAlOF and NAl IR.
  • Oligonucleotide NA13R is an alternative reverse primer.
  • reaction is set up as a one-step procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps.
  • the thermal cycle was programmed so that cDNA synthesis is followed immediately by PCR amplification, as follows:
  • the NA primers are The NA primers:
  • the reaction is set up as a one-step procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps. It is important to notice that this procedure is set up for mastercyclers with a ramping temperature of at least 4 °C/s for heating and 3 °C/s for cooling (the kit used was from Invitrogen).
  • the one-step RT-PCR reaction is as follows:
  • the thermal cycler program can be set up for 36 cycles instead of 43 cycles for the final amplification.
  • influenza forward primer 'NA8F' 5'- G[R]AC[H]CA[R]GA[R]TC[I][K][M][R]TG-3' SEQ ID NO: 9
  • influenza reverse primer 'NAlOR' 5'- CC[I][I][K]CCA[R]TT[R]TC[Y]CT[R]CA-3' are incorporated into a double stranded PCR product.
  • the PCR experiments included negative control containing all the components of the reaction mixture except template RNA. The volume of the component omitted was replaced by an equivalent volume of ultra pure distilled water DNAse and RNAse free
  • the agarose gel illustrates the re-amplification of cDNA obtained from a gel purified
  • sample typing Flu A/FluB
  • Matrix gene specific primer known to amplify only Flu A by QHPS.
  • a HA subtyping was performed by QHPS lab using Real time PCR based on HA specific primers
  • NA inhibition test was performed by a reference laboratory using c NA serum Antibody test was performed by a reference laboratory using
  • Taq Polymerase (5 U/ ⁇ l) 0.2 ⁇ l cDNA template X ⁇ l ( ⁇ 6 ng in total) ddH 2 O (DNAse/RNAse Free) to a final volume of 50 ⁇ l
  • DNAse/RNAse free water to a final volume of 50 ⁇ l
  • the PCR experiments included negative controls containing all the components of the reaction mixture except template DNA, and primers, since different pairs of primers were tested at the same time.
  • the volume of the components omitted was replaced by an equivalent volume of ultra pure distilled water DNAse and RNAse free.
  • the final PCR reaction samples are visualised by UV transilumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 2OmM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes.
  • DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100L, GeneWorks) that is used as a base pair size reference.

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Abstract

The present invention relates to a method of diagnosing influenza type A infection in an individual, the method comprising obtaining a biological sample, suspected of containing influenza virus particles, and optionally isolating genetic material from the sample; amplifying a segment of at least one gene selected from HA, NA and PB2 using a set of oligonulceotides, wherein the segment of the at least one gene contains a target sequence that is predictive of pathogenicity, virulence or drug resistance; and analysing the amplified target sequence.

Description

TITLE OF THE INVENTION
"DETECTION OF INFLUENZA VIRUS"
FIELD OF THE INVENTION
[0001] This invention relates generally to a method for analyzing a target nucleic acid sequence. More particularly, the present invention relates to a method for determining the presence or absence of influenza virus type A, or nucleic acid sequences derived therefrom, in a subject. Methods of the invention facilitate the amplification of segments of the influenza type A haemagglutinin (HA), neuraminidase (NA) and PB2 genes from all variant type A subtypes using one set of oligonucleotides for each gene. Methods of the invention further enable the determination of the pathogenicity and/or virulence status of those particular strains/subtypes of Influenza type A detected in accordance with the invention through analyzing the amplified segments which contain a specific target sequence(s). The present invention further relates to kits for use in accordance with methods of the invention to amplify and analyze target nucleic acid sequences for the detection of influenza virus type A and all its subtypes.
BACKGROUND OF THE INVENTION
[0002] Influenza infection (flu) is transmitted by tiny droplets of moisture spread from the respiratory tract of infected individuals by coughing, sneezing or even talking. When these are breathed in by a susceptible individual the viruses they contain can enter the cells of the respiratory tract and multiply. The individual will usually become ill within 2-3 days. Two types of influenza viruses are of great public health concern; they are designated as influenza type A and influenza type B and can be readily distinguished from each other by laboratory tests but not by the clinical symptoms they produce. Influenza type A viruses are contracted by in excess of 100 million people worldwide each year and such viruses have been demonstrated to mutate into the highly pathogenic subtypes. In birds, influenza A
(H5N1) virus is a subtype of avian flu that is highly contagious and deadly. This viral strain does not usually infect humans, however, infections have been known to have occurred. The majority of cases relate to people in close contact with H5N1 infected poultry.
[0003] The surface of influenza viruses are covered with two types of glycoproteins, known as haemagglutinin (HA) and neuraminidase (NA), which, in electron microscope images, appear as a fringe or spikes projecting from the surface. The HA protein is the most abundant and is responsible for attachment of the virus to the host cell membrane, whilst the NA protein is a host cell receptor destroying enzyme, thereby facilitating release of progeny virions. Influenza type A is classified by its' subtypes. The subtypes are dependent on the sequences of the HA and NA surface proteins. Many different combinations of HA and NA proteins are possible, and each combination represents a different subtype. So far, 16 HA [H1-H16] and nine NA [N1-N9] subtypes have been detected in birds and mammals worldwide (Rohm et al, 1996, Virology, 217: 508-516).
[0004] Within one subtype, a spectrum from non-pathogenic to highly pathogenic strains may exist. High pathogenicity can primarily be determined by the presence of multiple basic amino acids at the cleavage site of the HA protein. The HA RNA is translated into a single precursor polypeptide, termed HAO, approximately 556 residues in length (Zambon, 1999, J. Antimicrob. Chemother, 44 (supp B): 3-9). To be infectious, HAO must be further cleaved into two peptides, HAl and HA2, linked together by a disulphide bridge (Webster and Rott, 1987, Cell, 50(5): 665-666). The cleavage is carried out by the hosts' proteases. In highly pathogenic strains, there are an increased number of basic residues at the cleavage site thought to arise from insertion or substitution. The increase in basic residues allows proteases present in tissues outside the gastro-intestinal and respiratory tract to cleave and activate the precursor polypeptide and hence render the virus infectious to a greater number of tissues (Yuen et al., 1998, Lancet, 351 (9101): 467-471). Mutations within the NA gene are associated with resistance of influenza type A to antiviral drugs. For example, mutations at codons corresponding to amino acid residues between positions 820 and 880 of the NA protein, determine resistance of influenza type A virus to Oseltamivir (Tamiflu).
[0005] The PB2 protein is a critical component of the influenza type A viral polymerase and is also associated with virulence. Approximately 75% of the highly virulent forms of H5N1 influenza virus isolates from Vietnam had a mutation consisting of a change to lysine at amino acid residue 627 in the PB2 protein. Mutations at amino acid residue 355 have been associated with the ability of the virus to replicate in humans.
[0006] Influenza type A is usually detected through the use of conventional diagnostic tools, cell culture and serologic testing which can take between two and fourteen days for results. In a disease outbreak situation, such testing procedures would not allow for the prevention of such a virulent infection from spreading throughout a population. Although, more efficient methods of virus detection have been discovered, a reliable and rapid method for the detection of all virus subtypes is still being investigated. [0007] At present, there are a range of methods by which the influenza virus can be analysed to determine virulence and pathogenicity. The first being a classical method of isolating haemagglutin and/or neuraminidase and antigenically characterising them by inhibition tests using specific antisera against the 16 HA known subtypes and nine NA known subtypes. Rapid assays have been designed for the detection of viral antigen in tissue impression smears and cryostat sections by use of immunofluorescence, or by antigen-capture enzyme-linked immunosorbent assays (ELISA) (Cattoli et al., 2004, Avain Pathol, 33(4): 432-437).
[0008] Approaches incorporating molecular techniques are becoming increasingly popular. One such technique is based on the presence of influenza type A RNA in a sample, detected through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (Spackman et al., 2002, J. Clin. Microbiol, 40(9): 3256-3260). When influenza is detected, the amplified fragments of genes are analysed to characterise the specific subtypes. When the subtype is determined, the fragment can then be further analysed to establish the amino acid sequence at the endoproteolytic cleavage site, in turn indicating the pathology of the virus.
[0009] A similar method based on Real-Time RT-PCR has also been used to detect influenza virus. Enders et al., (2005, Emerging Infectious Diseases, 11(8): 1303-1305) developed a multiplex assay that employs a mixture of two sets of primers and dual labelled fluorescent probes that specifically target two different regions of the HA gene. Phipps et al., (2004, J. Virol. Methods, 122: 119-122) involved the use of a single set of primers for the amplification of a wide range of HA2 subtypes of influenza type A viruses.
[0010] Molecular amplification techniques represent a rapid and reliable means for determining the presence of influenza virus with the added advantage that many samples can be analysed by less personnel on a large scale. Furthermore, molecular amplification techniques and sample preparation can also be readily automated. However, a major problem faced in the design of oligonucleotide primers is the failure of the primers to successfully amplify regions within genes (for example the HA, NA and PB2 genes) of all subtypes of influenza type A. There are potentially 144 different combinations of HA and NA for any strain of influenza type A virus. The use of specific primers for each combination would neither be cost effective nor time effective. Furthermore, without analyzing all influenza type A subtypes, there can be no certainty of diagnosis, leading to the problem of false negative results. There is a clear need for the development of an efficient method for detecting the large number of influenza type A virus subtypes. [0011] The present invention is predicated in part on the unexpected discovery that diagnostically useful sequences in the HA, NA and PB2 genes of all influenza type A virus subtypes can be amplified through the use of a minimal set of specific oligonucleotide primers. The invention may be employed without prior knowledge of the precise sequence of the gene(s) and/or the virus subtype. Furthermore, the amplified sequences can be additionally analyzed to determine the pathogenicity of the virus, thereby providing an extremely rapid and sensitive method for influenza A diagnosis.
SUMMARY OF THE INVENTION [0012] In a first aspect, the present invention provides a method for detecting subtypes or strains of influenza type A virus, the method comprising: analyzing a segment of at least one gene selected from HA, NA and PB2 using a plurality of oligonucleotides, wherein at least one segment contains a target sequence that is predictive of pathogenicity, virulence or drug resistance. [0013] In a second aspect, the present invention provides a method of diagnosing influenza type A infection in an individual, the method comprising:
- obtaining a biological sample, suspected of containing influenza virus particles, and optionally isolating genetic material from the sample;
- amplifying a segment of at least one gene selected from HA, NA and PB2, wherein an individual segment contains a target sequence that is predictive of pathogenicity, virulence or drug resistance; and
- analyzing the amplified target sequence.
[0014] In a third aspect, the present invention provides a test kit suitable for the detection of influenza type A from a biological sample, comprising: - oligonucleotides for amplifying a segment of at least one gene selected from
HA, NA and PB2, wherein an individual segment contains a target sequence that is predictive of pathogenicity, virulence or drug resistance; and
- reagents for nucleic acid amplification, including at least one reagent selected from, pure water, sterile tubes, hot wax, filter tips, buffer and wash solution. [0015] In one embodiment, the present invention provides a method for amplifying a target nucleic acid sequence in a test sample suspected of containing influenza type A virus. This method generally comprises:
- combining in a reaction vessel on ice: 1. DNA from a biological sample, comprising of but not restricted to blood, bodily fluids, faeces, sputum, saliva and tissue biopsy;
2. an oligonuclotide set capable of annealing to at least one, two or three genes selected from HA, NA and PB2, wherein the individual segment(s) to be amplified contains a target sequence that is predictive of pathogenicity, virulence or drug resistance; 3. the appropriate concentration and quantity of reagents required for any suitable amplification method known in the art; subjecting the contents of the reaction vessel to a nucleic acid processing reaction to form one or more reaction products if the target nucleic acid is present in the test sample, wherein the reaction product(s) thus formed comprise the predictive target sequence; and
- detecting the reaction product(s) through the use of techniques known in the art such as agarose gel electrophoresis, hybridization, direct sequencing, which indicate the presence of influenza A virus and determining the size, identity and/or quantity of the nucleic acid sequence. [0016] The method may include the purification of RNA from the sample and the subsequent reverse transcription of the RNA to cDNA. Accordingly, the method of amplification may be by reverse transcriptase PCR using the cDNA so produced as template.
[0017] The amplification reaction may incorporate one, two or even three gene products amplified in the same reaction vessel. Alternatively, amplification reactions for the detection of HA, NA and PB2 gene segments may be carried out individually in separate reaction vessels, with specific amplification protocols.
[0018] The amplification techniques may include but are not restricted to PCR, Reverse Transcriptase PCR, Real-time PCR, Real-time Reverse Transcriptase PCR, strand displacement amplification, rolling circle amplification, nucleic acid bound amplification (NASBA), ligase chain reaction and QB replicase amplification. [0019] Methods of the invention may be used to determine the pathogenicity and virulence status of the particular strain/subtype of Influenza type A through analyzing the genomic segments, which contain a specific target sequence. Such techniques include but are not limited to the use of nucleotide probes, restriction endonuclease digest, Nested PCR and DNA sequencing.
[0020] The target sequence of the HA gene may encompass codons corresponding to amino acid residues at positions 226 and 228 of the HA protein, which are predictive for binding to receptors on avian cells or human cells. Alternatively, or in addition, the target sequence comprises the protease cleavage site within the HA gene responsible for cleaving the HAl and HA2 polypeptides. In an embodiment, the oligonucleotides specific for the HA gene are capable of detecting all known HA subtypes. The oligonucleotides may comprise the sequences as set forth in any of SEQ ID NOs: 5 to 7 and 18 to 19.
[0021] The target sequence of the NA gene may encompass one or more codons corresponding to amino acid residues between positions 820 to 880 of the NA protein, in which mutations occur that determine resistance to Oseltamivir (Tamiflu). In an embodiment, the oligonucleotides specific for the NA gene are capable of detecting all known NA subtypes. The oligonucleotides may comprise the sequences as set forth in any of SEQ ID NOs: 9 to 17 and 20 to 22.
[0022] The target sequence of the PB2 gene may encompass a codon corresponding to amino acid residue at position 627 and/or position 355 of the PB2 protein, which are predictive of replication efficiency of the virus, and associated with the ability of the virus to infect and replicate in humans, respectively. The oligonucleotides may comprise the sequences as set forth in any of SEQ ED NOs: 1, 2, 3 and 4.
[0023] In another embodiment, the present invention further relates to a kit that can be used in accordance with methods of the invention to amplify and analyze a target nucleic acid sequence for the detection of Influenza Virus Type A and all its subtypes. The kit may contain the oligonucleotides of the invention and reagents for completing amplification techniques, preferably PCR. The kit may also contain instructions on volumes of reagents and amplification methods. [0024] The aspects and embodiments of the invention apply to any mammalian or avian species susceptible to infection by the influenza type A virus. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure l is a photographic representation of an agarose gel showing a 986 bp PB2 gene segment generated by one-step RT-PCR using forward primer 'PB2(1)F' (SEQ ID NO: 1) and reverse primer 'PB2(2)R' (SEQ ID NO: 2). Agarose gel marker, lOObp Hyperladder II (Bioline Pty Ltd), Lane 1; Negative control, water in place of template RNA, Lane 2; lOμl of PCR product amplified from the M+ gene segment of Influenza A using H5N3 as a template, Lane 3; lOμl of PCR product using H5N3 viral RNA as template, Lane 4; lOμl of PCR product using Hl 1N6 viral RNA as template, Lane 5; lOμl of PCR product using H7N7 viral RNA as template, Lane 6; lOμl of PCR product using H12N9 viral RNA as template, Lane 7; lOμl of PCR product using H7N7 viral RNA as template, Lane 8; lOμl of PCR product using H4N4 viral RNA as template, Lane 9; lOμl of PCR product using H6N5 viral RNA as template, Lane 10; lOμL of PCR product using H9N2 viral RNA as template, and Lane 11; Negative control, water in place of template DNA as in Lane 2. Lanes 1-6, half the usual amount of superscript III (lμl) was used. [0026] Figure 2 is a photographic representation of an agarose gel showing a
589bp PB2 gene segment generated by one-step RT-PCR using forward primer 'PB2(1)F' (SEQ ID NO: 1) and reverse primer 'PB2(1)R' (SEQ ID NO: 3). Agarose gel marker, lOObp Hyperladder II (Bioline Pty Ltd); Lane 1, Negative control, water in place of template RNA; Lane 2, lOμl of PCR product amplified from the M gene segment of Influenza A using H5N3; Lane 3, lOμl of PCR product using H5N3 viral RNA as template; Lane 4, lOμl of PCR product using Hl 1N6 viral RNA as template; Lane 5, missed sample; Lane 6, lOμl of PCR product using H12N9 viral RNA as template; Lane 7, lOμl of PCR product using H7N7 viral RNA as template; Lane 8, lOμl of PCR product using H4N4 viral RNA as template; Lane 9, lOμl of PCR product using H6N5 viral RNA as template; Lane 10, lOμl of PCR product using H7N4 viral RNA as template; and Lane 11, lOμl of PCR product using H7N4 viral RNA as template. All lanes used half the usual amount of superscript III (lμl).
[0027] Figure 3 is a photographic representation of an agarose gel showing a 395bp PB2 gene segment generated by one-step RT-PCR using forward primer 'PB2(2)F' (SEQ ID NO: 4) and reverse primer 'PB2(2)R' (SEQ ID NO: 2). Agarose gel marker, lOObp Hyperladder II (Bioline Pty Ltd); Lane 1, Negative control, water in place of template RNA; Lane 2, lOμl of PCR product amplified from the M gene segment of Influenza A using H5N3; Lane 3, lOμl of PCR product using H5N3 viral RNA as template; Lane 4, lOμl of PCR product using Hl 1N6 viral RNA as template;, Lane 5, lOμL of PCR product using H7N7 viral RNA as template; Lane 6, lOμl of PCR product using H12N9 viral RNA as template; Lane 7, lOμl of PCR product using H7N7 viral RNA as template; Lane 8, lOμl of PCR product using H4N4 viral RNA as template; Lane 9, lOμl of PCR product using H6N5 viral RNA as template; Lane 10, lOμl of PCR product using H7N4 viral RNA as template; and Lane 11, lOμl of PCR product using H7N4 viral RNA as template. All lanes used half the usual amount of superscript III (lμl).
[0028] Figure 4 is a photographic representation of an agarose gel showing a 395bp or 589 bp (approx.) products from PB2 gene segment generated by one-step RT-PCR using forward primer 'PB2(2)F' (SEQ ID NO: 4) and reverse primer 'PB2(2)R' (SEQ ID NO: 2) to generate the 395 bp product; or the PB2(1)F (SEQ ID NO: 1) and PB2(1)R (SEQ ID NO: 3) to generate the 589 bp product. Agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 14, lOμl of PCR product using H7N7 viral RNA as template; Lane 13, lOμl of PCR product using H5N3 viral RNA as template; Lane 12, lOμl of PCR product using H7N4 viral RNA as template; Lane 11, lOμl of PCR product using H7N4 viral RNA as template and amplified by primers complementary to the M gene segment (as a positive control); Lane 10, lOμl of PCR product using H7N4 viral RNA as template and amplified using primers complementary to the M gene segment (as a positive control) ; Lane 9, lOμl of PCR product using H5N1 viral RNA as template. Lane 8, negative control, water in place of template; Lane 7, lOμl of PCR product using H7N7 viral RNA as template; Lane 6, lOμl of PCR product using H5N3 viral RNA as template; Lane 5, lOμl of PCR product using H7N4 viral RNA as template; Lane 4, lOμl of PCR product using H3N2 viral RNA as template; Lane 3, lOμl of PCR product using H7N3 viral RNA as template; Lane 2, lOμl of PCR product using H9N2 viral RNA as template; Lane 1, lOμl of PCR product using H5N1 viral RNA as template. Lanes marked 'large M' and 'small M' are amplified from the M gene segment of influenza type A. All other lanes are amplified using the PB2 primer set. Lanes 1 to 8 are using primers PB2(2)F (SEQ ID NO: 4) and PB2(2)R (SEQ ID NO: 2). Lanes 9, 12, 13 and 14 are using primers PB2(1)F (SEQ ID NO: 1) and PB2(1)R (SEQ ID NO: 3).
[0029] Figure 5 is a photographic representation of an agarose gel showing a 986 bp PB2 gene segment generated by two-step RT-PCR using reverse primer 'PB2(2)R' (SEQ ID NO: 2) for cDNA synthesis; and using forward primer 'PB2(1)F' (SEQ ID NO: 1) and reverse primer 'PB2(2)R (SEQ ID NO: 2) to amplify the cDNA. Lane M: agarose gel marker, 100 bp Hyperladder II (Bioline Pty Ltd); Lane 1, 10 μL of PCR product using H1N9 viral RNA as template; Lane 2, lOμl of PCR product using H3N8 viral RNA as template; Lane 3, lOμl of PCR product using H5N3 viral RNA as template; Lane 4, 10 μl of PCR product using H6N5 viral RNA as template; Lane 5, lOμl of PCR product using H7N7 viral RNA as template; Lane 6, lOμl of PCR product using Hl 1N6 viral RNA as template; Lane 7, lOμl of PCR product using H12N9 viral RNA as template and; Lane 8, lOμl of PCR product using H15N9 viral RNA as template.
[0030] Figure 6 is a photographic representation of an agarose gel showing a 500 bp Haemagglutin gene segment generated by two-step RT-PCR using primer 'Unil2' (SEQ ID NO: 8) for cDNA synthesis; and forward primer 'RLHAP03F (SEQ ID NO: 5) and reverse primer 'RLHAP09R' (SEQ ID NO: 6) to amplify the cDNA. Agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lOμl of PCR product using H3N8 viral cDNA as template; Lane 2, lOμl of PCR product using H7N7 viral cDNA as template; Lane 3, lOμl of PCR product using H6N5 viral cDNA as template; Lane 4, lOμl of PCR product using H1N9 viral cDNA as template; Lane 5, lOμl of PCR product using Hl 1N6 viral cDNA as template and; Lane 6, lOμl of PCR product using H12N9 viral cDNA as template.
[0031] Figure 7 is a photographic representation of an agarose gel showing a 500 bp Haemagglutinin gene segment generated by two-step RT-PCR using primer 'Unil2' (SEQ ID NO: 8) for cDNA synthesis; and forward primer 'RLHAP03F (SEQ ID NO: 5) and reverse primer 'RLHAP09R' (SEQ ID NO: 6) to amplify cDNA. Agarose gel marker,
Hyperladder II (Bioline Pty Ltd); Lane 1, Negative control, water in place of template DNA; Lane 2, Positive control, amplification of a NA gene segment generated by two-step RT-PCR using primer 'Unil2' (SEQ ID NO: 8) for cDNA synthesis; and forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR1 (SEQ ID NO: 12) to amplify cDNA. The primer combination 8F/10R gives a product of 219 bp; Lane 3, lOμl of HA gene segment PCR product using H5N3 viral cDNA as template; and Lane 4, lOμl of HA gene segment PCR product using H15N9 viral cDNA as template.
[0032] Figure 8 is a photographic representation of an agarose gel showing a 500bp Haemagglutinin gene segment generated by one-step RT-PCR using RLHAP03F (SEQ ID NO: 5) and reverse primer either RLHAP09MR (SEQ ID NO: 18) or RLHAP 1 OMR (SEQ ID NO: 19). Lane M: agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lOμl of HA gene segment RT-PCR product using H4N4 viral RNA as template and RLHAP03F/RLHAP09MR primers; Lane 2, lOμl of HA gene segment using H4N4 viral RNA as template and RLHAP03F/RLHAP10MR primers; Lane 3, 10 μl of negative control, water in place of template RNA and using RLHAP03F/RLHAP09MR primers. Lane 4, lOμl of negative control, water in place of template RNA and using RLHAP03F/RLHAP10MR primers.
[0033] Figure 9 is a photographic representation of an agarose gel showing a 219bp or 353 bp (approx.) products from Neuraminidase gene segment generated by two-step RT-PCR using primer 'NAl 3R' (SEQ LD NO: 16) for cDNA synthesis; and using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12) to generate the 219 bp product; or the 'NAlOF' (SEQ ID NO: 11) and 'NAl IR' (SEQ ID NO: 10) to generate the 353 bp product. The gel illustrates the amplification of gene fragments from five different subtypes and seven different viruses. Lane MM: agarose gel marker, DMW-100L (GeneWorks); Lane 2, lOμl of PCR product using H1N9 viral RNA as template; Lane 3, lOμl of PCR product using H1N9 viral RNA as template; Lane 5, lOμl of PCR product using H3N8 viral RNA as template; Lane 6, 1 Oμl of PCR product using H3N8 viral RNA as template; Lane 8, lOμl of PCR product using H5N3 viral RNA as template; Lane 9, lOμl of PCR product using H5N3 viral RNA as template, Lane 11, 1 Oμl of PCR product using H6N5 viral RNA as template; Lane 12, lOμl of PCR product using H6N5 viral RNA as template; Lane 14, lOμl of PCR product using H7N7 viral RNA as template; Lane 15, 1 Oμl of PCR product using H7N7 viral RNA as template; Lane 17, lOμl of PCR product using H12N9 viral RNA as template; Lane 18, lOμl of PCR product using H12N9 viral RNA as template; Lane 20, lOμl of PCR product using H15N9 viral RNA as template; Lane 21, lOμl of PCR product using H15N9 viral RNA as template.
[0034] Figure 10 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12). The gel illustrates the re- amplification of cDNA obtained from a gel purified PCR product from Figure 9. Agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lOμl of re-amp lified PCR product using H1N9 viral gel purified cDNA as template; Lane 2, lOμl of re-amplified PCR product using H3N8 viral gel purified cDNA as template; Lane 3, lOμl of re-amplified PCR product using H5N3 viral gel purified cDNA as template; Lane 4, lOμl of PCR product using H6N5 viral gel purified cDNA as template; Lane 5, lOμl of PCR product using H7N7 viral gel purified cDNA as template; Lane 6, lOμl of PCR product using H12N9 viral gel purified cDNA as template; Lane 7, lOμl of PCR product using H15N9 viral gel purified cDNA as template; Lane 8, Negative control, water in place of template DNA.
[0035] Figure 11 is a photographic representation of an agarose gel showing a 353 bp Neuraminidase gene segment generated by PCR using forward primer 'NAlOF' (SEQ ID NO: 11) and reverse primer 'NAl IR' (SEQ ID NO: 10). The gel illustrates the re- amplification of cDNA obtained from gel purified PCR product from Figure 9. Agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lOμl of re-amp lified PCR product using H3N8 viral gel purified cDNA as template; Lane 2, lOμl of re-amplified PCR product using H5N3 viral gel purified cDNA as template; Lane 3, lOμl of re-amplified PCR product using H6N5 viral gel purified cDNA as template; Lane 4, lOμl of re-amplified PCR product using H7N7 viral gel purified cDNA as template; Lane 5, lOμl of re-amplified PCR product using H12N9 viral gel purified cDNA as template.
[0036] Figure 12 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer
'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12). The gel illustrates the amplification of gene fragments from three different putative subtypes of viruses (e.g., N3, N9, N7). Lane MM: agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lOμl of PCR product using H5N3 viral RNA as template; Lane 2, lOμl of PCR product using H15N9 viral RNA as template; Lane 3, lOμl of PCR product using H7N7 viral RNA as template; Lane 4, Negative control, water in place of template RNA.
[0037] Figure 13 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12). The gel illustrates the amplification of gene fragments from three different putative subtypes of viruses (e.g., N4, N9 (previously unknown), Nl). Lane 1, lOμl of PCR product using H4N4 viral RNA as template; Lane 2, lOμl of PCR product using unknown viral RNA as template; Lane 3, lOμl of PCR product using H5N1 V viral RNA as template; Lane 4, Negative control, water in place of template RNA; Lane MM: agarose gel marker, Hyperladder II (Bioline Pty Ltd). [0038] Figure 14 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12). The gel illustrates the amplification of gene fragments from 3 different putative subtypes of viruses (e.g., N7, N4, Nl). Lane MM: agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 1, lOμl of PCR product using H7N7 viral RNA as template; Lane 2, lOμl of PCR product using H7N4 viral RNA as template; Lane 3, lOμl of PCR product using H5N1C viral RNA as template and; Lane 4, Negative control, water in place of template RNA.
[0039] Figure 15 is a photographic representation of an agarose gel showing a 219 bp Neuraminidase gene segment generated by one-step RT-PCR using forward primer 'NA8F' (SEQ ID NO: 9) and reverse primer 'NAlOR' (SEQ ID NO: 12). The gel illustrates the amplification of gene fragments from clinical samples. Lane H2O, Negative control, water in place of template RNA; Lane MM: agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 31, lOμl of PCR product using Influenza A sample 31 (H3N?, undetermined NA subtype) viral RNA as template; Lane 32, lOμl of PCR product using Influenza A sample No. 32 (H3N2 Wisconsin/67/2005 like strain) viral RNA as template; Lane 33, lOμl of PCR product using Influenza A sample No. 33 (H3N2) viral RNA as template; Lane 34. lOμl of PCR product using Influenza A sample No. 34 (H3N2) viral RNA as template; Lane 35, lOμl of PCR product using Influenza B sample No. 35 viral RNA as template, and serve as negative control; Lane 36, lOμl of PCR product using Influenza A sample 36 (H3N?, unknown NA subtype) viral RNA as template; Lane 37, lOμl of PCR product using Influenza A sample No. 37 (H3N2) viral RNA as template; Lane 8, lOμl of PCR product using Influenza A sample No. 8 (H3N2) viral RNA as template; Lane 38, lOμl of PCR product using Influenza A sample No. 38 (H3N2 Wisconsin/67/2005 like strain) viral RNA as template; Lane 39, lOμl of PCR product using Influenza B sample No. 39 viral RNA as template, and serve as negative control; Lane 40, lOμL of PCR product using Influenza A sample No. 40 (unknown subtype) viral RNA as template; Lane 41, lOμl of PCR product using Influenza B sample No. 41 viral RNA as template, and serve as negative control; Lane 42, lOμl of PCR product using Influenza A sample No. 42 (H3N3 Wisconsin/67/2005 like strain) viral RNA as template
[0040] Figure 16 is a photographic representation of an agarose gel showing a 253 bp (approximate) product from a Neuraminidase gene segment generated by PCR using forward primer 'NA8F-M13' (SEQ ID NO: 20) and reverse primer 'NA10R-M13' (SEQ ID NO: 21). The gel illustrates the re-amplification of cDNA obtained from a gel purified PCR product of clinical samples. Lane MM: agarose gel marker, Hyperladder II (Bioline Pty Ltd); Lane 21, lOμl of PCR product using Influenza A sample No. 21 (H3N?, unknown NA subtype) viral cDNA as template; Lane 30, lOμl of PCR product using Influenza A sample No. 30 (H3N2 Wisconsin/67/2005 like strain) viral cDNA as template; Lane 31, lOμl of PCR product using Influenza A sample No. 31 (H3N?, unknown NA subtype) viral cDNA as template; Lane 32, 10 μl of PCR product using Influenza A sample No. 32 (H3N2 Wisconsin/67/2005 like strain) viral cDNA as template; Lane 33, lOμl of PCR product using Influenza A sample No. 33 (H3N2) viral cDNA as template; Lane 34, lOμl of PCR product using Influenza A sample No. 34 (H3N2) viral cDNA as template; Lane 37, lOμl of PCR product using Influenza A sample No. 37 (H3N2) viral cDNA as template; Lane 38, lOμl of PCR product using Influenza A sample No. 38 (H3N2 Wisconsin/67/2005 like strain) viral cDNA as template; Lane H2O, Negative control, water in place of template DNA.
[0041] Figure 17 is a schematic representation of a phylogenetic tree showing the position of the sequences obtained from several samples from Table 3.
BRIEF DESCRIPTION OF THE SEQUENCES
Figure imgf000015_0001
The standard mix base definitions for the above listed oligonucleotide sequences of the present invention are as follows:
R = A, G Y = C,T M = A, C K = G5T S = C5G
W = A5T H = A5C5T B = C5G5T V = A5C5G D = A5G5T
I = inosine DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[0043] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
[0044] The terms "amplification" or "nucleic acid amplification" or "amplification reaction" refer to a biochemical reaction that produces many polynucleotide copies of a particular target nucleic acid sequence. If the target nucleic acid sequence is single-stranded complementary sequences may be produced in the reaction. In some embodiments, the reaction is a polymerase chain reaction (PCR) or a similar reaction that uses a polymerase to copy a nucleic acid sequence such as helicase dependent amplification (HDA), transcription mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA) and reverse transcription polymerase chain reaction (RT-PCR). A double stranded region formed through the hybridization of an oligonucleotide to a single-stranded form of the target nucleic acid sequence is required to prime (start) the reaction. In other embodiments, the terms "amplification" or "nucleic acid amplification" or "amplification reaction" refer to a biochemical reaction using a ligase or similar enzyme that covalently links two oligonucleotides or two oligonucleotide sub-sequences, such as a ligase chain reaction (LCR). Ligase enzymes ligate the two oligonucleotides or oligonucleotide sub-sequences when they hybridize at adjacent sites in the target nucleic acid sequence. Alternatively, if the two oligonucleotides or oligonucleotide subsequences hybridize at sites that are one or more nucleic acid residues apart, i.e., they are not adjacent, then the single stranded region between the double stranded regions is converted to a double stranded region using a polymerase, and the ligase enzyme then links the adjacent oligonucleotides to form a continuous double stranded region. [0045] The term "capturable sequence" refers to a nucleic acid sequence that is capable of hybridizing with another nucleic acid sequence.
[0046] The term "capture oligonucleotide array" means a plurality of capture oligonucleotides immobilized at discrete known locations on a solid surface, or on beads/colloids in suspension. In relation to the surface of a reaction vessel or diagnostic strip, the capture oligonucleotides may be arranged in a two-dimensional spatially addressed array, e.g., a 2 x 2 array. Alternatively, the capture oligonucleotides may be arranged in a tubular array in which a two-dimensional planar sheet is rolled into a three-dimensional tubular configuration. In other embodiments, the capture oligonucleotides is arranged on the inner or outer surface of a two- or three-dimensional reaction vessel of any convenient topology.
[0047] The terms "codon" and "codons" refer to a sequence of three adjacent nucleotides constituting the genetic code that specifies the insertion of an amino acid in a specific structural position in a polypeptide chain during the synthesis of proteins.
[0048] The terms "complementary" and "complementarity" refer to a sequence of nucleotides related by the base-pairing rules. For example, the sequence "A-G-T-C" is complementary to the sequence "T-C-A-G". Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules.
Alternatively, there may be "complete" or "total" complementarity between the nucleic acids.
The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
[0049] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. [0050] "Haemagglutinin" (HA) is an antigenic glycoprotein found on the surface of the influenza virus and is responsible for binding the virus to the cell that is being infected. The name haemagglutinin is given because the spikes are capable of adhering to red blood cells, causing them to be agglutinated.
[0051] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA, DNA-RNA or a DNA-PNA hybrid.
Complementary base sequences are those sequences that are related by the base-pairing rules. In relation to DNA, A pairs with T and C pairs with G. In relation to RNA, U pairs with A and C pairs with G. The base inosine (I) may also be used. Inosine can form base pairs with C or A or G or T (in descending order of stability). In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
[0052] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated oligonucleotide", as used herein, refers to an oligonucleotide, which has been purified from the sequences that flank it in a naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
[0053] "Influenza Type A genes" include but are not limited to the hemagglutinins Hl, H2, and H3; the neuraminidase gene, which encodes for example, Nl and N2; the NP gene, which encodes the nucleoprotein; the M gene, which encodes the matrix proteins; the NS gene, which encodes two different non-structural proteins and one RNA molecule (PA, PBI and PB2) for each of the three subunits of the RNA polymerase.
[0054] "Neuraminidase (NA)" is an antigenic glycoprotein enzyme found on the surface of the Influenza virus. The enzyme forms a mushroom-shaped projection on the surface of an influenza virus particle, and assists in the release of newly- formed virus particles from the surface of an infected cell.
[0055] The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotides and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule may vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotides. Oligonucleotides can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences. Not all bases in the oligonucleotide need reflect the sequence of the template molecule to which the oligonucleotide will hybridize, the oligonucleotide need only contain sufficient complementary bases to enable hybridization to the template. An oligonucleotide may also include mismatch bases at one or more positions, being bases that are not complementary to bases in the template, but rather are designed to incorporate changes into the DNA upon base extension or amplification. An oligonucleotide may include additional bases, for example in the form of a restriction enzyme recognition sequence at the 5 ' end, to facilitate cloning of the amplified DNA.
[0056] The terms "oligonucleotide", "polynucleotide" or "nucleic acid" as used herein designate DNA, cDNA, RNA, mRNA, cRNA or PNA. The terms "polynucleotide" and "nucleic acid" typically refers to oligonucleotides greater than 30 nucleotides in length.
[0057] The term "pathogenicity" refers to the ability of the influenza A virus agent of known virulence to produce disease in a range of hosts under a range of environmental conditions. [0058] The "PB2" gene encodes the PB2 protein which is a critical component of the viral polymerase of influenza.
[0059] By "primer" is meant an oligonucleotide which, when paired with a strand of DNA or RNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is typically single-stranded for maximum efficiency in amplification but may alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotides, although it may contain fewer nucleotides. Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more. Primers may be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary", it is meant that the primer is sufficiently complementary to hybridize with a target nucleotide sequence. Suitably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotides may be attached to the 51 end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotides or a stretch of non-complementary nucleotides can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer. In certain contexts throughout the specification, the terms "primer" and "oligonucleotide" may be used interchangeably.
[0060] Terms used to describe sequence relationships between two or more nucleic acid sequences include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 10 but frequently 15 to 20 and often at least 25 monomer units, i.e., nucleotides, in length. Because two nucleic acid sequences may each comprise: (1) a sequence (i.e., only a portion of the complete nucleotide sequence) that is similar between the two polynucleotides and; (2) a sequence that is divergent between the two nucleic acid sequences, sequence comparisons between two (or more) nucleic acid sequences are typically performed by comparing sequences of the nucleic acid sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 50 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et ai, (1997, Nucl. Acids Res. 25: 3389). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15. [0061] The terms "restriction endonuclease" and "restriction enzyme" refer to a group of enzymes which cut DNA at a specific site called the recognition site. They are endonucleases in that they cut within a DNA molecule rather than cutting at the ends. There are three types of restriction endonucleases, and the majority of recognition sites at which they cut are palidromic.
[0062] The terms "sequence identity" and "identity" are used interchangeably herein to refer to the extent that nucleic acid sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
[0063] The term "sequencing" refers to the method of determining a DNA sequence through the process of establishing the nucleotide order of a given DNA fragment, called the DNA sequence. Currently, almost all DNA sequencing is performed using the chain termination method, developed by Frederick Sanger. This technique uses sequence- specific termination of an in vitro DNA synthesis reaction using modified nucleotide substrates. [0064] The term "target sequence" is used herein to refer to any nucleic acid or amino acid sequence of interest. It may be an entire gene, or portion thereof. As such, the target nucleic acid sequence may be a potion of a gene comprising a genetic mutation such as, but not limited to, nucleotide insertions, deletions and single nucleotide polymorphisms (SNPs). The target nucleic acid sequence may also be a nucleic acid encoded by an entire gene, or portion thereof. The target nucleic acid sequences contemplated by the present invention include, therefore, DNA, cDNA, RNA, mRNA and cRNA. The target nucleic acid sequence may be mutated or contain an altered nucleic acid sequence. [0065] The term "strain" as used here in refers to an influenza A virus strain classified as low pathogenic (LPAI) or highly pathogenic (HPAI) on the basis of specific molecular genetic and pathogenic criteria that requires specific testing as described by the present invention. For example Avian influenza A viruses of the subtypes H5 and H7, including H5N1, H7N7, and H7N3 viruses, have been associated with HPAI, and human infection with these viruses have ranged from mild (H7N3, H7N7) to severe and fatal disease (H7N7, H5N1).
[0066] "Stringency" as used herein, refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the degree of complementarity between hybridized nucleic acid sequences.
[0067] "Stringent conditions" as used herein refers to temperature and ionic conditions under which only polynucleotides and oligonucleotides that are substantially complementary or having a high proportion of complementary bases, preferably having exact complementarity, will hybridize and, in some embodiments, yield amplification products. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used. Stringent conditions are well known to those of skill in the art. Generally, for oligonucleotides used as probes in hybridization reactions stringent conditions are selected to be about 10 to 20°C less than the calculated thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe. Tm calculations are well known to those expert in the art. Tm is best calculated for oligonucleotides less than 14 base residues long by using the formulae Tm = 4(wG + xC) + 2(yA + zT) -16.6*logl0(0.050) + 16.6*loglO([Na+]), where w, x, y, z are the number of the bases G, C, A, T in the sequence, respectively, and [Na+] is the salt concentration. For oligonucleotides longer than 13 base residues, the Tm is calculated by the nearest neighbour formulae as described by Breslauer et al., (1986, Proc. Nat. Acad. Sci. 83: 3746-50), but using the values published by Sugimoto et al, (1996, Nucl. Acids Res. 24: 4501-4505). Values for RNA thermodynamic properties can be taken from Xia et al., (1998, Biochemistry, 37: 14719-14735). It will be understood that an oligonucleotide probe or primer will hybridize to a target sequence under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions. Reference herein to low stringency conditions for probe hybridization reactions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 420C, and at least about 1 M to at least about 2 M salt for washing at 42°C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 rnM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 niM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions for probe hybridization reactions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42°C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42°C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 650C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 42°C. High stringency conditions for probe hybridization reactions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at a temperature in excess of 650C. Other stringent conditions for probe hybridization reactions are well known in the art. A skilled addressee will recognise that various factors can be manipulated to optimise the specificity of the hybridization. Optimisation of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Current protocols in Molecular Biology {supra) at pages 2.10.1 to 2.10.16 and Molecular Cloning, a Laboratory Manual, (Sambrook, et ai, eds. Cold Spring Harbor Press 1989) at sections 1.101 to 1.104.
[0068] The terms "subtype" and "subtypes" refer to groups of influenza type A viruses, wherein a subtype is classified on the basis of the identity of one or more proteins on the surface of the virus, typically the HA and NA proteins. Examples of such subtypes of influenza A viruses found in people are A(HlNl) and A(H3N2). Influenza B virus is not divided into subtypes.
[0069] The term "virulence" refers to the ability of an infectious agent to do damage to the host. Viral virulence factors determine whether the infection occurs and how severe the resulting viral disease symptoms are. Virulence can be determined on a scale which ranges from high to low.
2. Methods for analyzing a target nucleic acid sequence
2.1 Genetic material [0070] The present invention is directed to molecular methods and kits for determining the presence of influenza virus type A in a subject and to methods for amplifying target nucleic acid sequences derived from influenza virus type A. Embodiments of the invention facilitate the analysis and amplification of segments of the influenza type A haemagglutinin (HA) genes, the neuraminidase (NA) genes and PB2 genes from all variant subtypes using at least one oligonucleotide primer for each gene. Advantageously, embodiments of the invention may be employed without prior knowledge of the exact sequence of the gene(s) in question or of the strain/subtype of the virus present in the sample to be tested. Those skilled in the art will appreciate that the methods and kits of the present invention may be used alone or in conjunction with other available testing procedures in identifying influenza type A viruses, viral particles and nucleic acid sequences derived there from. Embodiments of the invention facilitate, for the first time, the detection and analysis of all known HA and NA subtypes of influenza type A virus.
[0071] As described an exemplified herein, analysis of segments of the influenza type A genes for the detection of influenza virus subtypes in accordance with the invention may be carried out by amplification, such as polymerase chain reaction. However, it will be readily appreciated by those skilled in the art that analysis may be carried out using a variety of techniques for sequence analysis known in the art, with or without prior amplification, without departing from the scope of the present invention.
[0072] In accordance with embodiments of the invention, any source of nucleic acid, in purified or non-purified form, can be utilized as the starting nucleic acid or acids, provided it contains or is suspected of containing the specific nucleic acid sequence desired. Thus, the process may employ, for example, DNA or RNA, including mRNA, which DNA or RNA may be single stranded or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of any of these nucleic acids may also be employed, or the nucleic acid produced from a previous amplification reaction herein using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture. The starting nucleic acid may contain more than one desired specific nucleic acid sequence which may be the same or different. [0073] The nucleic acid(s) to be amplified may be obtained from any cell source or body fluid. Non- limiting examples of cell sources include blood cells, or any cells present in tissue obtained by biopsy. Body fluids include, blood, saliva, urine, faeces, cerebrospinal fluid, semen and tissue exudates at the site of infection or inflammation. Nucleic acids may be extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract the nucleic acids will depend on the nature of the source. Typically the nucleic acids to be analysed are isolated from the biological sample to be tested. However, it will be readily appreciated by those skilled in the art that methods of the invention may be carried out without prior nucleic acid isolation using techniques known in the art. [0074] In practicing methods of the present invention, an RNA, DNA or cDNA sample is typically contacted with pairs of oligonucleotide primers, under conditions suitable for the amplification of the DNA or cDNA. Typically, nucleic acid samples are amplified using polymerase chain reaction methods.
2.2 Oligonucleotides (primers) [0075] Oligonucleotides of the present invention hybridize to a target nucleic acid sequence when it is present in a test sample. Simultaneous extension of the annealed primers from a 3' terminus of each primer synthesizes an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the nucleic acid, if present, serves as a template for the synthesis of an extension product for the other primer of each pair; and analyzing the sample for the presence or absence of amplified products. The presence of amplified products indicates the presence of influenza A, or nucleic acids derived there from, in the sample.
[0076] Oligonucleotide primers may be provided in double-stranded or single- stranded form, although the single-stranded form is desirable. In certain embodiments, oligonucleotide primers are labelled with radioactive species 32P, 14C, 35S, 3H, or other label), with a fluorophore (e.g., rhodamine, fluorescein) or with a chemillumiscent label (e.g., luciferase). [0077] The optimal concentration for primers can be evaluated by performing single PCR reactions using each primer pair individually. For example, the concentration of each primer in the reaction mixture may range from about 0.01 pmol/μl to about 100 pmol/μl, or about 0.1 pmol/μl to about 10 pmol/μl, or about 0.2 pmol/μl to about 7 pmol/μl. The primer pair should be in the orientation that permits amplification. The forward primer should be in the 5'-3' orientation. The reverse primer should be in an inverse complementary orientation. One primer is designed to be complementary to the negative (-) strand and the other is complementary to the positive (+) strand. The primers should be chosen as such that they are not complementary at the 3' end. A complementary region of equal to or greater than two nucleotides will cause an unwanted primer hybridization. Preferably, there will be no complementary region at the 3' end. Also preferred are primers that do not have internal complementary segments that allow formation of hairpins. Typically, hybridization conditions utilizing at least two primer pairs of the invention include, for example, a hybridization temperature of about 50 0C to about 65 0C and a MgCl2 concentration of about 1.5 mM to about 2.0 mM. Although lower temperatures and higher concentrations OfMgCl2 can be employed, this may result in decreased primer specificity.
[0078] In an embodiment, the oligonucleotides should be at least 10 nucleotides in length. The oligonucleotides should amplify the desired region(s) of DNA or cDNA which contains the specified signature/target sequence. Annealing the primers to the denatured nucleic acid followed by extension with a suitable enzyme, and nucleotides results in newly synthesized + and - strands containing the target sequence. Suitable enzymes are any enzymes for use in extension and amplification reactions as will be well known to those skilled in the art including, but are not limited to, the large fragment of DNA Polymerase I (Klenow), Taq polymerase, Pfu polymerase and Pfx polymerase. Because these newly synthesized sequences are also templates for the primers, repeated cycles of denaturing, primer annealing and extension results in exponential accumulation of the region defined by the primer. The product of the chain reaction will be a discrete nucleic acid duplex with terminal corresponding to the ends of the specific primers employed.
[0079] Oligonucleotides of the invention include those suitable for the amplification of diagnostically useful sequences from the HA and NA genes and capable of detecting all known HA and NA subtypes of influenza type A virus. Such oligonucleotides include those exemplified herein, comprising the sequences as set forth in SEQ ID NOs: 5 to 7, and SEQ ID NOs: 18 to 19 (HA subtype-specific) and SEQ ID NOs: 9 to 17 and 20 to 22 (NA subtype-specific). Also exemplified herein are primers suitable for amplification of diagnostically useful sequences of the PB2 gene, as set forth in SEQ ID NOs: 1 to 4. However those skilled in the art will appreciate that the present invention is not limited to the use of the specific oligonucleotides exemplified, but alternative oligonucleotide sequences may also be used, provided the oligonucleotides are designed appropriately so as to enable the amplification of target sequences from the HA, NA and/or PB2 genes. For example, in alternative embodiments, the nucleotide sequence of a suitable oligonucleotide may share at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity with the sequence of an oligonucleotide as exemplified herein. Those skilled in the art will appreciate that one or base substitutions, additions or deletions of these sequences may be made in generating a oligonucleotide of at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity.
[0080] In an embodiment, the oligonucleotides of the invention for the amplification of segments of the influenza type A haemagglutinin (HA), neuraminidase (NA) and PB2 genes from type A subtypes, can be chimerised with other sequences, for example sequencing primers (e.g., M13F and M13R) or tags for capture onto solid substrates.
2.3 Amplification methods
[0081] Nucleic acids used in methods of the invention can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al, 1989, supra; and Ausubel et al, 1994, supra). The nucleic acid is typically fractionated (e.g., poly A+ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the influenza type A gene segments present in a given template sample.
[0082] Representative methods for nucleic acid amplification are well known in the art, and include, but are not limited to, PCR (see, e.g., Saiki et al, 1985, Science, 230: 1350-1354; Mullis et al, 1987, Methods Enzymol, 155: 335-350), Strand Displacement Amplification (SDA and multiple SDA (MSDA); see, e.g., US Patent No. 5,422,252 and Little et al.,) Rolling Circle Amplification (RCA; see, e.g., Liu et al., 1996, J. Am. Chem. Soc, 118: 1587-1594 and US Patent No. 5,854,033 and US Patent No. 6,642,034), Nucleic Acid Sequence Based Amplification (NASBA; see, e.g., Sooknanan et al, 1994, Biotechniques, 17: 1077-1080), Ligase Chain Reaction (LCR; see, e.g., WO 89/09835) and Qβ Replicase Amplification (see, e.g., Tyagi et al, 1996, supra). Various permutations on these techniques may also be employed as reviewed by Syvanen Anne-Christine, (2001, supra). For example, the PCR may be reverse transcriptase PCR (RT-PCR), Real-Time PCR or Real-Time RT-PCR. In the present method, any useful combination of features of different amplification reactions may be used to increase the sensitivity and/or specificity of the method.
[0083] An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, Ausubel et al, {supra), and in Innis et al, ("PCR Protocols", Academic Press, Inc., San Diego Calif, 1990). Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If a cognate influenza type A gene sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated. A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, (1989, supra). Alternative methods for reverse transcription utilise thermostable, RNA- dependent DNA polymerases. These methods are described in WO 90/07641 and polymerase chain reaction methodologies are well known in the art. [0084] In certain advantageous embodiments, the template-dependent amplification involves the quantification of transcripts in real-time. For example, RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al, Biotechnology, 10: 413-417). By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesised from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA.
[0085] Another method for amplification is the ligase chain reaction ("LCR"), disclosed in European Patent Application No. 320 308. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
[0086] Qβ Replicase, described in PCT Application No. PCT/US87/00880, may also be used. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.
[0087] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'α- thio-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al., (1992, Proc. Natl. Acad. Sci. U.S.A, 89: 392-396).
[0088] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.
[0089] Still another amplification method described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, may be used. In the former application, "modified" primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labelled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.
[0090] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Proc. Natl. Acad. ScL U.S. A, 86:1173; Gingeras et al, PCT Application WO 88/10315). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerisation, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerisation. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.
[0091] Davey et al, EPO No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double- stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
[0092] Miller et al., in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, M. A., In: "PCR Protocols: A Guide to Methods and Applications", Academic Press, N.Y., 1990; Ohara et al, 1989, Proc. Natl Acad. Sd. U.S.A, 86: 5673-567).
[0093] In other embodiments, the polymerase dependent amplification comprises rolling circle amplification (RCA), in which hybridization of oligonucleotide primers to a circular nucleic acid molecule permits ligation, i.e., circularization, and a DNA polymerase, typically one that has strand displacement activity, to synthesise a first extension product using the circular nucleic acid molecule as a template. Generally, the extension product is a long nucleic acid molecule containing multiple repeats of sequences complementary to the template circular nucleic acid molecule. In the presence of a complementary oligonucleotide primer, the first extension product can then serve as a template for the synthesis of further extension products, apropos of PCR, thereby permitting amplification of the original template circular nucleic acid molecule.
[0094] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used for amplifying target nucleic acid sequences. Wu et al., (1989, Genomics, 4: 560). [0095] Depending on the format, the influenza type A nucleic acid of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means (e.g., ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, J Macromol. Sci. Pure. Appl. Chem, A31(l):1355-1376). [0096] hi some embodiments, amplification products or "amplicons" are visualized in order to confirm amplification of the influenza type A gene sequences. One typical visualisation method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labelled with radio- or fiuorometrically-labelled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. In some embodiments, visualization is achieved indirectly. Following separation of amplification products, a labelled nucleic acid probe is brought into contact with the amplified influenza type A sequence. The probe is suitably conjugated to a chromophore but may be radiolabeled. Alternatively, the probe is conjugated to a binding partner, such as an antigen- binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule. The techniques involved are well known to those of skill in the art and can be found in many standard texts on molecular protocols (e.g., see Sambrook et al., 1989, supra and Ausubel et al., 1994, supra). For example, chromophore or radiolabel probes or primers identify the target during or following amplification.
3. Amplified target sequence analysis
3.1 Sequence detection techniques
[0097] The oligonucleotide sets of the present invention have been designed to amplify diagnostically useful sequences within the HA, NA and PB2 influenza type A genes. The HA target region encompasses codons that correspond to amino acid residues which are predictive for binding to receptors on avian cells or human cells. The target region also includes the HA cleavage site which determines the pathogenicity of influenza A viruses. The NA target sequence encompasses the region in which occur mutations that determine resistance to Oseltamivir (Tamiflu). The PB2 target sequence encompasses the region in which a mutation occurs in humans and birds consisting of an amino acid change to lysine or glutamic acid at position 627. To determine the sequence at each of these target regions a number of techniques may be utilised as described below. [0098] In one embodiment following separation of amplification products, a labelled nucleic acid probe is brought into contact with the amplified influenza type A gene segment sequence. The probe is suitably conjugated to a chromophore but may be radiolabeled. Alternatively, the probe is conjugated to a binding partner, such as an antigen- binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule. The techniques involved are well known to those of skill in the art and can be found in many standard texts on molecular protocols (e.g., see Sambrook et al., 1989, supra and Ausubel et al., 1994, supra). For example, chromophore or radiolabel probes or primers identify the target during or following amplification.
[0099] In certain embodiments, target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilised on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labelled) under conditions that promote denaturation and rehybridisation. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
[0100] Following detection/quantification, one may compare the results seen in a given subject with a control reaction or a statistically significant reference group of normal subjects or of subjects lacking influenza type A.
[0101] Also contemplated are genotyping methods and allelic discrimination methods and technologies such as those described by Kristensen et al., (2001, Biotechniques, 30(2): 318-322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqMan®, liquid chromatography, and mass spectrometry. [0102] Also contemplated for the analysis of gene segments amplified in accordance with the invention are biochip-based technologies such as those described by Hacia et al., (1996, Nature Genetics, 14: 441-447) and Shoemaker et al, (1996, Nature Genetics, 14: 450-456). By tagging amplified gene segments with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridisation. See also Pease et al, (1994, Proc. Natl. Acad. ScL U.S.A, 91: 5022-5026); Fodor et al., (1991, Science, 251: 767- 773). Briefly, nucleic acid probes to influenza type A polynucleotides are made and attached to biochips. The nucleic acid probes attached to the biochip are designed to be substantially complementary to specific influenza type A gene segments amplified or the target sequence contained therein or to other probe sequences, (for example in sandwich assays), such that hybridisation of the target sequence and the probes of the present invention occurs. This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridisation between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridisation can occur under even the least stringent of hybridisation conditions, the sequence is not a complementary target sequence. In certain embodiments, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e., have some sequence in common), or separate.
[0103] As will be appreciated by those of ordinary skill in the art, nucleic acids can be attached to or immobilised on a solid support in a wide variety of ways. By "immobilised" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non- covalent. By "non-covalent binding" and grammatical equivalents herein is meant one or more of either, electrostatic, hydrophilic, and hydrophobic interactions. Included in non- covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilisation may also involve a combination of covalent and non-covalent interactions.
[0104] In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesised first, with subsequent attachment to the biochip, or can be directly synthesised on the biochip.
[0105] The biochip comprises a suitable solid or semi-solid substrate or solid support. By "substrate" or "solid support" is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by practitioners in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalised glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc. In general, the substrates allow optical detection and do not appreciably fluoresce.
[0106] Generally the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well. For example, the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimise sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
[0107] In certain embodiments, oligonucleotide probes are synthesised on the substrate, as is known in the art. For example, photoactivation techniques utilising photopolymerisation compounds and techniques can be used. In an illustrative example, the nucleic acids are synthesised in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505 and U.S. Pat. NOs. 5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis of the Affymetrix GeneChip technology. [0108] In an illustrative biochip analysis, oligonucleotide probes on the biochip are exposed to or contacted with amplified segments of one or more influenza type A genes under conditions favouring specific hybridisation.
[0109] cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases. Suitably, cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilised oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridisation conditions. Alternatively, fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers. [0110] The target Influenza type A polynucleotides can be detectably labelled so that their hybridisation to individual probes can be determined. The target polynucleotides are typically detectably labelled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu34), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide, hi some embodiments in which an enzyme is used as a direct visual label, biotinylated bases are incorporated into a target polynucleotide. Hybridisation is detected by incubation with streptavidin-reporter molecules.
[0111] Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al., (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al.,) 5,326,692 (Brinkley et al.,). Alternatively, reference may be made to the fluorochromes described in U.S. Patent NOs. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially available fluorescent labels include, for example, fluorescein phosphoramidites such as Fluoreprime™ (Pharmacia), Fluoredite (Millipore) and FAM (Applied Biosystems International) [0112] Radioactive reporter molecules include, for example, 32P, which can be detected by an X-ray or phosphorimager techniques.
[0113] The hybrid- forming step can be performed under suitable conditions for hybridising oligonucleotide probes to test nucleic acid including DNA or RNA. In this regard, reference may be made, for example, to Nucleic Acid Hybridisation, A Practical
Approach (Homes and Higgins, Eds, IRL press, Washington D. C, 1985). In general, whether hybridisation takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid- forming region, the viscosity of the medium and the possible presence of denaturants. Such variables also influence the time required for hybridisation. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation.
[0114] In certain advantageous embodiments, high discrimination hybridisation conditions are used. For example, reference may be made to Wallace et al., (1979, Nucl. Acids Res. 6: 3543) who describe conditions that differentiate the hybridisation of 11 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch. Reference also may be made to Wood et al., (1985, Proc. Natl. Acid. Sci. USA, 82: 1585) who describe conditions for hybridisation of 11 to 20 base long oligonucleotides using 3 M tetramethyl ammonium chloride wherein the melting point of the hybrid depends only on the length of the oligonucleotide probe, regardless of its GC content. In addition, Drmanac et al., {supra) describe hybridisation conditions that allow stringent hybridisation of 6-10 nucleotide long oligomers, and similar conditions may be obtained most readily by using nucleotide analogues such as 'locked nucleic acids (Christensen et al., 2001, Biochem J, 354: 4S1-4).
[0115] Generally, a hybridisation reaction can be performed in the presence of a hybridisation buffer that optionally includes a hybridisation optimising agent, such as an isostabilising agent, a denaturing agent and/or a renaturation accelerant. Examples of isostabilising agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts. Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulphoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts. Hybridisation accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol. Hybridisation buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM. [0116] A hybridisation mixture containing the target influenza type A marker polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridisation between the target sequences in the amplified polynucleotide segments and any complementary probes. Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound. Generally, incubation will be at temperatures normally used for hybridisation of nucleic acids, for example, between about 20 0C and about 75 0C, example, about 25 0C, about 30 0C, about 35 0C, about 400C, about 45 0C, about 50 0C, about 55 0C, about 60 0C, or about 65 0C. For probes longer than 14 nucleotides, 20 0C to 50 0C is desirable. For shorter probes, lower temperatures are preferred. A sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridisation between the target sequences in the target polynucleotides and any complementary probes. For example, the hybridisation may be carried out at about 45 0C +/- 10 0C in formamide for 1-2 days.
[0117] After the hybrid-forming step, the probes are washed to remove any unbound nucleic acid with a hybridisation buffer, which can typically comprise a hybridisation optimising agent in the same range of concentrations as for the hybridisation step. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridised to a target polynucleotide.
[0118] The hybridisation reactions are then detected to determine which of the probes has hybridised to a corresponding target sequence. Depending on the nature of the reporter molecule associated with a target polynucleotide, a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a coloured colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light. In such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe: target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymatically generated colour spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array. In the case of a nucleic acid array, the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In certain embodiments, oligonucleotide probes specific for different influenza type A gene sequences are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a
'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung et al., (U.S. Patent No. 5,143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al., (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labelled microbeads, the reaction may be detected using flow cytometry.
[0119] In certain embodiments, target sequences of the invention may be determined through the use of restriction endonucleases. Restriction endonucleases used under specific conditions which are well known in the art may be efficient in digesting the target DNA sequences of the highly pathogenic strains of influenza as opposed to the low pathogenic strains, or vice versa.
[0120] Suitably, the target sequences of the invention may also be determined through the use of nested PCR. Nested PCR is the method by which two pairs of PCR primers are used for the amplification of a single locus. The first pair amplify the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first PCR product and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.
4. Kits
[0121] All the essential components required for amplifying and analysing a target nucleic acid sequence in a test sample according to methods of the present invention may be assembled together in suitable kit form. Kits of the invention may optionally include appropriate components including but not restricted to, positive and negative controls, dilution buffers and the like. Also included may be components suitable for subjecting nucleic acids to a nucleic acid processing and/or amplifying reaction. These components include various polymerases such as, but not limited to, Taq polymerase, reverse transcriptase, DNA ligase etc. (depending on the nucleic acid processing reaction technique employed), nucleotide precursors, salts and buffer solutions. Kits may further include reagents for carrying out analysis of the amplified nucleic acids, such as an appropriate restriction enzyme(s), reaction buffer(s) for restriction enzyme digestion, and reagents for use in separating digested fragments (e.g., agarose) and/or nucleic acid sequencing. Kits of the invention may also comprise distinct containers for the individual components, hi some embodiments, the kits comprise instructions for performing the methods of the present invention.
[0122] In a particularly preferred embodiment, the kit of the present invention comprises a reaction vessel having HA, NA and PB2 oligonucleotides immobilized thereto and containing a pre-prepared mixture of reagents, suitably in lyophilized form.
[0123] hi order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.
EXAMPLES
EXAMPLE 1
RT-PCR DETECTION OF THE INFLUENZA A POLYMERASE GENE SEGMENT PB2
RT-PCR Primer desisn: [0124] RT-PCR primers were designed to amplify a part of the PB2 segment from a wide range of subtypes of influenza A virus.
[0125] The PB2 influenza forward primer PB2(1)F comprises the sequence 5'- AG[Y]TC[I]TC[Y]TT[Y]AG[Y]TT[Y]GG - 3' (SEQ ID NO: 1). The PB2 influenza reverse primer PB2(2)R comprises the sequence 5'- AGTAT[Y]CTCAT[Y]CC[W]GANCC -3' (SEQ ID NO: 2). The expected size of the product generated from these primers is approximately 986bp (refer to Figure 1). However, this may vary depending upon the viral isolate present in a sample of interest as the length of the PB2 coding sequence may vary slightly between isolates.
[0126] Additional primer sequences were designed to amplify smaller sections of the PB2 gene segment. Still using PB2(1)F as the forward primer, the additional reverse primer PB2(1)R comprises the sequence 5'- C[I]GG[I]GA[Y]A[R][K]AG[Y]A[Y] [R]TT[Y]C -3' (SEQ ID NO: 3). The expected size of the product generated from the above primer pair is approximately 589 bp (refer to Figure 2). However, this may vary depending upon the viral isolate present in a sample. [0127] An additional forward primer was designed, PB2(2)F which comprises the sequence 5'- GA[I]GT[I]AG[Y]GA[R]AC[M]CA[R]GG -3' (SEQ ID NO: 4). The expected size of the product generated from the primer pair PB2(2)F and PB2(2)R (SEQ ID NO: 2) is approximately 395bp (refer to Figure 3). However, this may also vary depending upon the viral isolate present in a sample.
Extraction of RNA from a sample:
[0128] Viral RNA was extracted from the sample (e.g., amniotic fluid or clinical sample) using a QIAGEN RNA easy extraction kit by following the manufacturer's instructions. 100 μl of sample was inactivated by addition of 600 μl of a guanidium denaturant and 6 μl of 2-mercaptoethanol prior to use in the QIAGEN extraction protocol. The extracted RNA was resuspended in 50 μl of RNase free water (QIAGEN) and 2 μl of the resuspended RNA was used in the RT-PCR reaction.
The " two step" RT-PCR reaction, cDNA synthesis followed by PCR:
[0129] cDNA synthesis:
1. Reverse Transcription mix
• 100μM PB2(2)R Primer 2μl
• RNA 5μl
■ dNTP mix 2μl ■ dH2O 4μJ
Total Reaction 13μl
Denaturation: 2. Heat mixture at 650C for 5 minutes
3. Put mixture on ice for 1 minute
4. Preparation reaction mixture (Invitrogen reagents)
■ 5X RT Buffer 4μl
■ 0. IM DTT lμl ■ RNAse Out lμl
■ Superscript™ III lμl
Add the preparation mix to the reverse transcription total reaction mix
Total Reaction 20μl
5. Place reaction at 42°C for 60 minutes
6. Heat inactivate reaction at 70°C for 15 minutes
[0130] PB2 Specific PCR Amplification
1. PCR reaction mixture:
1OX PCR Buffer 6μl
5OmM MgCl2 2μl
1OmM dNTP 2μl lOOμM selected PB2(1) 2μl
100μM PB2(2)R 2μl Platinum Taq Polymerase 0.2μl
• DNA template lμl
■ ClH2O 34.8μl Total Reaction 50μl
2. Add the following to a 0.5 ml, nuclease-free, thin- walled PCR tube on ice:
■ 1 OxPCR Buffer 5μl ■ Template DNA 1 μl or serial dilutions of the control plasmid
(as quantification standards) (2 μl)
Sense primer selected PB2 primer 2 μl (200 pmoles) Anti-sense primer selected PB2 primer 2 μl (200 pmoles) Platinum Taq Polymerase Mix (Invitrogen) 0.2 μl (5 U/μl) ■ 5OmM MgCl2 (2μl)
■ 10 mM dNTP (2μl) RNAse free water to 50 μl
3. Gently mix and centrifuge briefly to ensure that all the components are at the bottom of the amplification tube. Place the reaction plate or tubes in the preheated thermal cycler programmed as described below:
Denaturation:
• 1 cycle: 94°C for 2 min PCR amplification: ■ 8 cycles of step down PCR consisting of:
■ 15 s denaturation at 940C
• 30s annealing at 56°C, 54°C, 520C, 5O0C, 48°C, 46°C,44°C, 42°C in each of the successive "step down cycles"
• 75s extension at 68°C followed by:
36 cycles:
■ 94°C for 15s (denature)
■ 400C for 30 s (anneal)
■ 68°C for 75s (extend) Final extension (optional):
■ 1 cycle: 72°C for 5 min • Hold at 11 °C until required
4. As the reaction proceeds, the influenza PB2 forward primers and the influenza PB2 reverse primers are incorporated into a double stranded PCR product.
[0131] The final PCR reaction sample is visualised by UV transillumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 2OmM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes. DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100 L, GeneWorks) that is used as a base pair size reference.
[0132] The above protocol was successfully used to detect PB2 gene segments from a variety of influenza type A virus isolates, as illustrated in Figure 5.
[0133] The reaction is alternatively set up as a one-step procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps.
One-Step RT-PCR and PCR reaction: [0134] The one step RT-PCR reaction protocol for the PB2 gene segment:
The thermal cycler was programmed so that cDNA synthesis is followed immediately by PCR amplification, as follows:
The PB2 primers:
PB2(1)-F 5'-AGYTCITCYTTYAGYTTYGG-S' (SEQ ID NO: 1) PB2(1)-R 5'-CIGGIGAYARKAGYAYRTTYC-S' (SEQ ID NO:3)
N may be substituted for "I" in these above primer sequences and the primers generate an approximate 589 bp product (refer to Figure 2).
PB2(2)-F 5'-GAIGTIAGYGARACMCARGG-S ' (SEQ ID NO:4)
N may be substituted for "I" in this above primer sequence. PB2(2)-R 5 '-AGTATYCTCATYCCWGANCC-S ' (SEQ ID NO: 2)
Generate an approx. 395 bp product (refer to Figure 3).
The combination of PB2(1)F and PB2(2)R generates an approx. 986 bp product (refer to Figure 1).
[0135] The reaction is set up as a 'one-step' procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps. Note the different temperature for extension (as this is carried out at the temperature recommended for the superscript enzyme rather than the Taq used in the two step procedure (below)).
1. PCR reaction mixture:
25 μL superscript III 2X reaction mix (contains 0.4 mM each dNTP, 3.2mM MgSO4)
■ 2μL of forward primer (PB2(1)F, 16OuM solution) • 2μL of reverse primer (PB2(2)R, 16OuM solution) 2 μL Superscript III enzyme
■ 17μL RNase free H2O
2μL template RNA in RNAse free water (added last)
Total reaction volume = 50μl
2. Program for thermal cycler:
46°C 30 min
600C 10 min
3. PCR cycle:
94 °C 2 min
94 °C 15sec
56 °C 30sec
68 °C 1 min 15 sec
94 °C 15 sec
54 °C 30sec
68 °C 1 min 15 sec
94 °C 15 sec
52 °C 30sec 68°C 1 mini 5 sec
94°C 15 sec
50°C 30sec
68°C 1 mini 5 sec
94°C 15 sec
48°C 30sec
68°C 1 mini 5 sec
94°C 15 sec
46°C 30sec
68°C Iminl5sec
94°C 15 sec
44°C 30sec
68°C Iminl5sec
94°C 15 sec
42°C 30sec
68°C 1 mini 5 sec
94°C 15 sec
40°C 30sec
68°C 1 mini 5 sec
Repeat for 36 cycles
68°C 5min
[0136] For the long (approx. 986 bp) PB2 product a 75 sec extension time was used (as above). For the shorter products generated using the combination of primers PB2(1) F and PB2(1) R (approx 589 bp) or PB2(2)F and PB2(2) R (approx 395 bp) a shorter extension time of 40 sec was used.
[0137] The above protocol was successfully used to detect PB2 gene segments from a variety of influenza type A virus isolates, as illustrated in Figures 1-4.
EXAMPLE 2
RT-PCR DETECTION OF THE INFLUENZA A POLYMERASE GENE SEGMENT HA
RT-PCR Primer design:
[0138] RT-PCR primers are designed to amplify a part of the HA segment from a wide range of subtypes of influenza A virus.
[0139] The HA influenza forward primer RLHAP03F comprises the sequence 5'- T[I]TGGGG[I][R]T[I][M]A[Y]CA[Y][Y]C-3' (SEQ ID NO: 5). The HA influenza reverse primer RLHAP09R comprises the sequence 5'- CCA[I]CCA[I]CC[I][Y][Y][Y]TC -3' (SEQ ID NO: 6). The expected size of the product generated from these primers is approximately 500 bp. However, this may vary depending upon which virus is present in a sample of interest as the length of the HA coding sequence may vary slightly between isolates. (An alternative reverse primer to RLHAP09R (RL(2)HAP09R) comprises the sequence 5'- CCI[K][I]CCA [I]CC[I] [Y][Y][Y]TC -3' (SEQ ID NO: 7). In addition, two reverse primers have been designed, which comprise the sequence of 5'-CC[I][K][I]CCA[I]CC[I][Y][Y][Y]TC[I]AT-3 '(SEQ ID NO: 18) and 5'-A[I][I]CC[I][K][I]CCA[I]CC[I][B][Y][Y]TC-3' (SEQ ID NO: 19).
Extraction of RNA from a sample:
[0140] Viral RNA was extracted from the sample (e.g., amniotic fluid or clinical sample) using a QIAGEN RNA Easy extraction kit by following the manufacturer's instructions. 100 μl of sample was inactivated by addition of 600 μl of a guanidium denaturant and 6 μl of 2-mercaptoethanol prior to use in the QIAGEN extraction protocol. The extracted RNA was resuspended in 50 μl of RNAse free water (QIAGEN). 2 μl of the resuspended RNA was used in the RT-PCR reaction.
The Two-step RT-PCR reaction: [0141] cDNA synthesis:
1. Reverse Transcription mix • lOμM Uni 12 Primer 2μl
■ RNA 5μl
■ dNTP mix 2μl
■ dH2O 4μJ Total Reaction 13μl
The Unil2 primer sequence is 5'-AGCAAAAGCAGG-S' (SEQ ID NO: 8). Denaturation:
2. Heat mixture at 65 °C for 5 minutes
3. Put mixture on ice for 1 minute
4. Preparation reaction mixture (Invitrogen reagents) • 5X RT Buffer 4μl
■ 0. IM DTT lμl RNAse Out lμl
■ Superscript™ III lμl
Add the preparation mix to the reverse transcription total reaction mix
Total Reaction 20μl
5. Place reaction at 420C for 60 minutes
6. Heat inactivate reaction at 70°C for 15 minutes [0142] HA Specific PCR Amplification 1. PCR reaction mixture:
• 1OX PCR Buffer 5μl ■ 5OmM MgCl2 2μl
• 1OmM dNTP lμl
• 100μM RLHAP03F primer 0.75μl
■ 100μM RLHAP09R HA primer 0.75μl
• Platinum Taq Polymerase (5U/μL) 0.5μl ■ DNA template 4μJ
• dH2O 36JiI
Total Reaction 50μl
2. Add the following to a 0.2 ml, nuclease-free, thin-walled PCR tube on ice:
1 OxPCR buffer (Invitrogen) 5μl
Template DNA 4 μl or serial dilutions of the control plasmid (as quantification standards) (2 μl) • Sense primer RLHAP03F 0.75 μl (75 pmoles)
Anti-sense primer RLHAP09R 0.75 μl (75 pmoles) Platinum Taq Polymerase Mix (Invitrogen) 0.5 μl (5 U/μl)
• 50 rnM MgCl2 (2μl) lO mM dNTP's (lμl) • RNAse free water to 50 μl 3. Gently mix and centrifuge briefly to ensure that all the components are at the bottom of the amplification tube. Place the reaction plate or tubes in the preheated thermal cycler programmed as described below:
Denaturation: ■ 1 cycle: 94°C for 5 min
PCR amplification:
8 cycles of step down PCR consisting of: 30s denaturation at 94°C
■ 30s annealing at 56°C, 54°C, 520C, 50°C, 48°C, 46°C,44°C, 42°C in each of the successive "step down cycles"
75 s extension at 72°C followed by:
36 cycles: 94°C for 30s (denature) • 44°C for 30 s (anneal) • 72°C for 75s (extend)
Final extension (optional):
1 cycle: 720C for 5 min Hold at 110C until required
4. As the reaction proceeds, the influenza forward primer RLHAP03F
5'-T[l]TGGGGI[R]TI[M]ACA[Y][Y]C-3' (SEQ ID NO: 5) and the influenza reverse primer RLHAP09R 5'-CCA[I]CCA[I]CC[I][Y][Y][Y]TC -3' (SEQ ID NO: 6) are incorporated into a double stranded PCR product .
[0143] The final PCR reaction sample is visualised by UV transillumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 20 mM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes. DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100L, Gene Works) that is used as a base pair size reference. [0144] The above protocol was successfully used to detect HA gene segments from the following avian influenza type A virus isolates (see Table 1), as illustrated in Figures 6 and 7. TABLE 1:
Figure imgf000050_0001
[0145] The reaction is alternatively set up as a one-step procedure without the separate production of cDNA. This procedure is faster; requiring shorter cycle times and no manipulation between the RT and PCR steps (refer to Figure 8).
HA One-step RT-PCR
1. PCR reaction mixture:
• 2X RT-PCR Buffer 12.5μl 100 μM 3F HA forward primer 1 μl 100 μM 9R HA reverse primer 1 μl RT-PCR enzyme mixture 1 μl
■ DNA template lμl
■ dH2O 8.5ιιl
Total Reaction 25μl
2. RT-PCR cycles:
RT condition:
1 cycle: 460C for 30 min 1 cycle: 60°C for 10 min Denaturation:
1 cycle: 94°C for 3 min PCR amplification:
8 cycles of step down PCR consisting of: 30s denaturation at 94°C
• 30s annealing at 56°C, 540C, 52°C, 5O0C, 480C, 46°C,44°C, 42°C in each of the successive "step down cycles"
75s extension at 68°C Followed by:
36 cycles: 94°C for 30s (denature)
• 43°C for 30 s (anneal)
• 68°C for 75s (extend) Final extension (optional):
1 cycle: 68°C for 10 min Hold at 11 °C until required
EXAMPLE 3
RT-PCR DETECTION OF THE INFLUENZA A POLYMERASE GENE SEGMENT NA
RT-PCR Primer design:
[0146] RT-PCR primers were designed to amplify a segment of the NA gene from a wide range of subtypes of influenza A virus.
[0147] Two pairs of primers were designed for the amplification of the NA gene segment, these consist of:
1. The NA influenza forward primer 'NA8F' comprises the sequence 5'- G[R]AC[H]CA[R]GA[R]TC[I][K][M][R]TG -3' (SEQ ID NO: 9) and the NA influenza reverse primer 'NAlOR' comprises the sequence 5'- CC[I][I][K]CCA[R]TT[R]TCfY]CT[R]CA -3' (SEQ ID NO: 12). 2. The NA influenza forward primer 'NAlOF' comprises the sequence 5'-
TG[Y]AG[R]GA[Y]AA[Y]TGG[M][l][l]GG-3' (SEQ ID NO: 11) and the NA influenza reverse primer 'NAUR' comprises the sequence 5'- CC[D]A[S]A[R]TA[l]CC[l]GACCA[R]T-3' (SEQ ID NO: 10).
[0148] The expected size of the product generated from the 'NA8FVNA10R' primers is approximately 219 bp. The expected size of the product generated from the
'NAlOFVNAl IR' primers is approximately 353 bp. However, this may vary depending upon the viral isolate present in a sample of interest as the length of the NA coding sequence may vary slightly between isolates. Extraction of RNA from sample:
[0149] Viral RNA was extracted from the sample (e.g., amniotic fluid or clinical sample) using a QIAGEN RNA easy extraction kit (for the samples derived from amniotic fluid) or a Roche MagNA Pure LC total nucleic acid isolation kit (for clinical samples) by following the manufacturer's instructions.
[0150] For the samples derived from amniotic fluid, 100 μl of sample was inactivated by addition of 600 μl of a guanidium denaturant and 6 μl of 2-mercaptoethanol prior to use in the QIAGEN extraction protocol. The extracted RNA was resuspended in 50 μl of RNase free water (QIAGEN). 5 μl of the resuspended RNA was used in the two-step RT-PCR reaction and 2 μl of the resuspended RNA was used in the one-step RT-PCR reaction.
[0151] For the clinical samples, RNA was extracted from nasopharyngeal aspirates (NPA) specimens provided by the Molecular Diagnostic Unit of Queensland Health Pathology and Scientific Services (QHPSS) as blind specimens. The clinical specimens were collected from suspect cases of viral respiratory disease during a period of September to October 2006, mainly from Queensland (Australia) population. The initial NA subtyping described in Figures legends: 15 and 16 was done by QHPSS either by NA inhibition test or NA serum antibody. There are some NA subtypes unknown by these methods. The RNA was extracted from 200 μl of NPA samples using Roche MagNA Pure LC total nucleic acid isolation kit and RNA was eluted in 100 μl of elution buffer. 1 μl of the resuspended RNA was diluted in 1 μl of ultra pure water, and then the 2 μl total volume were used in the one- step RT-PCR reaction.
The Two-step RT-PCR reaction, cDNA synthesis followed by PCR: [0152] cDNA synthesis: 1. Reverse Transcription mix lOOμM 'NAl 3R' gene specific primer 2μl
• RNA 5μl
• dNTP mix (1OmM) lμl
• dH2O 5μJ
Total Reaction 13μl
Denaturation:
2. Heat mixture at 65°C for 5 minutes 3. Put mixture on ice for 1 minute
4. Preparation reaction mixture (Invitrogen reagents)
■ 5X RT Buffer 4μl
• 0. IM DTT lμl ■ RNAse Out (40 units/μl) lμl
• Superscript™ III lμl
Add the preparation mix to the reverse transcription total reaction mix
Total Reaction 20μl
5. Place reaction at 55°C for 60 minutes
6. Heat inactivate reaction at 720C for 15 minutes [0153] NA Specific PCR Amplification 1. PCR reaction mixture:
1OX PCR Buffer (no MgCl2) 5μl
5OmM MgCl2 2μl lOmM dNTP's lμl
100 μM 'NA8F' 2μl lOOμM 'NAlOR' 2μl
Taq Polymerase (5 U/μl) 0.2μl cDNA template 2μl ddH2O (RNAse Free) 35.80ul
Total Reaction 50μl
2. Add the following to a 0.2 ml, nuclease-free, thin-walled PCR tube on ice:
• 1 OXPCR Buffer 5 μl • 50 mM MgCl2 2μl
■ lO mM dNTP's lμl
• Sense primer 'NA8F' 2μl (200 pmoles)
■ Anti-sense primer 'NAlOR' 2μl (200 pmoles) Recombinant Taq Polymerase Mix (Invitrogen) 0.2μl (5 U/μl) ■ Template cDNA or serial dilutions of the control plasmid (as quantification standards) 2μl
■ DNAse/RNAse free water 35.80μl 3. Gently mix and centrifuge briefly to ensure that all the components are at the bottom of the amplification tube. Place the reaction plate or tubes in the preheated thermal cycler programmed as described below:
PCR cycle:
94°C for 3 min
940C 30sec
56°C 30sec
72°C 1 min 15 sec
94°C 30sec
54°C 30sec
720C 1 min 15 sec
94°C 30sec
52°C 30sec
72°C 1 min 15 sec
94°C 30sec
5O0C 30sec
720C 1 min 15 sec
94°C 30sec
48°C 30sec
72°C 1 min 15 sec
940C 30sec
46°C 30sec
72°C 1 min 15 sec
940C 30sec
44°C 30sec
72°C 1 min 15 sec
94°C 30sec
42°C 30sec
72°C 1 min 15 sec
94°C 30sec
43°C 30sec
72°C 1 min 15 sec
Repeat for 36 cycles
720C lOmin
4°C Hold
[0154] As the reaction proceeds, the influenza forward primer 'NA8F' 5'-
G[R]AC[H]CA[R]GA[R]TC[I][K][M][R]TG-3' (SEQ ID NO: 9) or 'NAlOF' 5'- TG[Y]AG[R]GA[Y]AA[Y]TGG[M][I][I]GG -3' (SEQ ID NO:11) and the influenza reverse primer 'NAlOR' 5'- CC[I][I][K]CCA[R]TT[R]TC[Y]CT[R]CA-3' (SEQ ID NO: 12) or 'NAl IR' 5'- CC[D]A[S]A[R]TA[I]CC[I]GACCA[R]T -3' (SEQ ID NO: 10) are incorporated into a double stranded PCR product.
[0155] The PCR experiments included negative controls containing all the components of the reaction mixture except template DNA, and primers, since different pairs of primers were tested at the same time. The volume of the components omitted was replaced by an equivalent volume of ultra pure distilled water DNAse and RNAse free.
[0156] The final PCR reaction samples are visualised by UV transilumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 2OmM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes. DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100L, Gene Works) that is used as a base pair size reference.
[0157] The above protocol was successfully used to detect NA gene segments from the following avian influenza type A virus isolates (see Table 2), as illustrated in Figures 9 to 11.
TABLE 2:
Figure imgf000055_0001
[0158] Products shown in Figures 9, 10, 12, 13, and 14 were cloned and sequenced by Sanger sequencing methods. The summary of sequencing results is shown in Table 3. A phylogenetic tree illustrated in Figure 17, was elaborated with some sequences obtained from Table 3.
[0159] Alternate oligonucleotide sequences for the amplification of target sequences within the NA gene have also been designed as follows: 'NA5F': 5'-CA[Y][D][S][I]AATGR[I]AC[M][R]T[I][M]A[I]GA-3' (SEQ ID NO: 13) 'NAI lF': 5'-A[Y]TGGTC[I]GG[I]TA[Y]T[S]T[H]GG-3' (SEQ ID NO: 14) 'NA8R': 5'-CA[Y][K][M][I]GA[Y]TC[Y]TG[D]GT[Y]C-3' (SEQ ID NO: 15) •NA13R': 5'-[K]G[I][W][M][I]T[K][S]C[M][I]GATGG[I][K]C-S1 (SEQ ID NO: 16) 'NA9R': 5'-[R]CA[D]GA[R]CA[Y]TC[Y]TC[S][N][N][R]TG-S1 (SEQ ID NO: 17)
[0160] The alternative oligonucleotide sequences (NA5F, NAl IF, NA8R, NA13R and NA9R) may be used in a variety of combinations, including NA8F and NAlOR, NA5F and NA8R, NA8F and NA9R and NAlOF and NAl IR. Oligonucleotide NA13R is an alternative reverse primer.
[0161] Alternatively, the reaction is set up as a one-step procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps.
TABLE 3:
Figure imgf000056_0001
The One step RT-PCR reaction protocol for the NA εene segment:
The thermal cycle was programmed so that cDNA synthesis is followed immediately by PCR amplification, as follows:
The NA primers:
'NA8F' 5'- G[R]AC[H]CA[R]GA[R]TC[l][K][M][R]TG-3' (SEQ ID NO: 9) 'NAlOR' 51- CC[l][l][K]CCA[R]TT[R]TC[Y]CT[R]CA-3' (SEQ ID NO: 12) The above primers generate approx. a 219 bp product
3. The reaction is set up as a one-step procedure without the separate production of cDNA. This procedure is faster, requiring shorter cycle times and no manipulation between the RT and PCR steps. It is important to notice that this procedure is set up for mastercyclers with a ramping temperature of at least 4 °C/s for heating and 3 °C/s for cooling (the kit used was from Invitrogen).
The one-step RT-PCR reaction is as follows:
Figure imgf000057_0001
Total reaction volume 50 μL
Program for thermal cycler:
46°C 30 min
60°C 10 min
94°C 3 min
94°C 30sec
56°C 30sec
68°C 1 min 15 sec
94°C 30sec
54°C 30sec
68°C 1 min 15 sec
940C 30sec
52°C 30sec
68°C 1 min 15 sec
94°C 30sec
50°C 30sec
68°C 1 min 15 sec
94°C 30sec
48°C 30sec
68°C 1 min 15 sec
94°C 30sec
46°C 30sec 68°C 1 mini 5 sec
94°C 30sec
44°C 30sec
680C 1 mini 5 sec
94°C 30sec
42°C 30sec
68°C 1 mini 5 sec
94°C 30sec
430C 30sec
680C Iminl5sec
Repeat for 43 cycles
680C lOmin
4°C Hold
[0162] Alternatively, the thermal cycler program can be set up for 36 cycles instead of 43 cycles for the final amplification.
[0163] As the reaction proceeds, the influenza forward primer 'NA8F' 5'- G[R]AC[H]CA[R]GA[R]TC[I][K][M][R]TG-3' (SEQ ID NO: 9) and the influenza reverse primer 'NAlOR' 5'- CC[I][I][K]CCA[R]TT[R]TC[Y]CT[R]CA-3' are incorporated into a double stranded PCR product. [0164] The PCR experiments included negative control containing all the components of the reaction mixture except template RNA. The volume of the component omitted was replaced by an equivalent volume of ultra pure distilled water DNAse and RNAse free
[0165] The final PCR reaction samples were visualized by UV transilumination after ethidium bromide staining in a 1.5% agarose gel buffered with IX tris-acetate (TAE) buffer (40 mM Tris, 20 mM glacial acetic acid, 10 mM EDTA [pH, 8.0], electrophoresed at 100 V for 40 min. DNA gels are run with a DNA molecular weight ladder Hyperladder II (Bioline Pty Ltd) that is used as a base pair size reference.
[0166] The above protocol was successfully used to detect NA gene segments from a variety of avian influenza type A virus isolates (derived from amniotic fluid), see Table 4 as illustrated in Figures 12 to 14. TABLE 4:
Strains used in this study
Influenza A/Chicken/Vietnam/8/04 (H5N1 V)
Influenza A/Chicken/Cambodia/ 1 A/04 (H5N1C)
Influenza A/Shearwater/ Aust/75 (H5N3)
Influenza A/Grey teal/W A/1762/79 (H4N4)
Influenza A/Emu/NSW/97 (H7N4)
Influenza A/Duck/Victoria/1/76 (H7N7)
Influenza A/Shelduck/WA/1762/79 (H15N9)
Influenza A/0901 (Unknown origin)
[0167] In addition, the one-step RT-PCR protocol was successfully used to detect NA gene segments from a variety of influenza type A virus isolates from clinical samples, as illustrated in Figure 15. However, this is just a small example of clinical samples ran. At present, 37 influenza type A samples were tested, 25 influenza B, and 1 Adenovirus. Some of the results obtained are as follows in Table 5 on page 60. Figure 16 illustrates a 253 bp product from a NA gene segment generated by PCR using SEQ ID NOs: 20 and 21 :
'NA8F-M13' 5'-GTAAAACGACGGCCAGTG[R]AC[H]CA [R]GA[R]TC[I][K][M][R]TG-S' 'NAl OR-Ml 3' 5'-CAGGAAACAGCTATGACCC[I][I][K]CCA[R]TT[R]TC[Y]CT[R]CA-S'
The agarose gel illustrates the re-amplification of cDNA obtained from a gel purified
PCR product of clinical samples.
[0168] PCR Products of clinical samples showed in Figure 15 were gel purified and used for the chimera PCR reaction. The results of the chimera PCR reaction are shown in Figure 16. The chimera PCR products were sequenced by the Sanger sequencing method. TABLE 5:
Figure imgf000060_0001
Table continued
Figure imgf000061_0001
'Clinical samples. The sample typing (Flu A/FluB) was performed using a Matrix gene specific primer known to amplify only Flu A by QHPS. a HA subtyping was performed by QHPS lab using Real time PCR based on HA specific primers b NA inhibition test was performed by a reference laboratory using c NA serum Antibody test was performed by a reference laboratory using
NVI = No virus identification
N/A1 = Subtype data not available
[0169] NA Chimera PCR Amplification 1. PCR reaction mixture:
1OX PCR Buffer (no MgCl2) 5μl
5OmM MgCl2 2μl lOmM dNTP's lμl
100 μM 'NA8F-M13' 2μl lOOμM 'NAl OR-Ml 3' 2μl
Taq Polymerase (5 U/μl) 0.2μl cDNA template X μl (~ 6 ng in total) ddH2O (DNAse/RNAse Free) to a final volume of 50μl
Total Reaction 50μl
2. Add the following to a 0.2mL, nuclease-free, thin-walled PCR tube on ice:
■ 1 OXPCR Buffer 5 μl • 50 mM MgCl2 2μl
• lO mM dNTP's lμl
■ Sense primer 'NA8F-M 13 ' 2μl (200 pmoles)
■ Anti-sense primer 'NAl OR-Ml 3' 2μl (200 pmoles) ■ Recombinant Taq Polymerase Mix (Invitrogen) 0.2μl (5U/μl)
Template cDNA 6 ng in total (the template was previously gel purified from the RT-PCR reaction)
DNAse/RNAse free water to a final volume of 50μl
3. Gently mix and centrifuge briefly to ensure that all the components are at the bottom of the amplification tube. Place the reaction plate or tubes in the preheated thermal cycler programmed as described below:
PCR cycle:
94°C 3 min
94°C 30sec
56°C 30sec
72°C 1 min 15 sec
94°C 30sec
54°C 30sec
72°C 1 min 15 sec
94°C 30sec
52°C 30sec
72°C 1 min 15 sec
94°C 30sec
50°C 30sec
72°C 1 min 15 sec
94°C 30sec
48°C 30sec
72°C 1 min 15 sec
94°C 30sec
46°C 30sec
72°C 1 min 15 sec
94°C 30sec
44°C 30sec
72°C 1 min 15 sec
94°C 30sec
42°C 30sec
72°C 1 min 15 sec
94°C 30sec
43°C 30sec 72°C 1 mini 5 sec Repeat for 30 cycles
720C lOmin
4°C 2 min
11°C Hold
[0170] The PCR experiments included negative controls containing all the components of the reaction mixture except template DNA, and primers, since different pairs of primers were tested at the same time. The volume of the components omitted was replaced by an equivalent volume of ultra pure distilled water DNAse and RNAse free.
[0171] The final PCR reaction samples are visualised by UV transilumination after ethidium bromide staining in a 1.5 % agarose gel buffered with IX tris-acetate (TAE) buffer (4OmM Tris, 2OmM glacial acetic acid, 1OmM EDTA [pH, 8.0], electrophoresed at 50 V for 1 hour and 30 minutes. DNA gels are run with a DNA molecular weight ladder (1 kb ladder DMW-100L, GeneWorks) that is used as a base pair size reference.
[0172] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
[0173] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.
[0174] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

CLAIMS:
1. A method for detecting subtypes or strains of influenza type A virus, the method comprising: i. analyzing a segment of at least one gene selected from haemagglutinin (HA), neuraminidase (NA) and PB2 using at least one oligonucleotide, wherein the segment of the at least one gene contains a target sequence that is predictive of pathogenicity, virulence or drug resistance.
2. The method of claim 1, wherein the gene segments are amplified.
3. The method according to claim 2, wherein individual segments of at least two of the HA, NA and PB2 genes are amplified.
4. The method according to claim 3, wherein respective segments of each of the HA, NA and PB2 genes are amplified.
5. The method according to claim 1, wherein the oligonucleotides are designed to detect multiple HA virus subtypes, multiple NA virus subtypes and/or multiple PB2 virus subtypes respectively.
6. The method according to claim 2 or 5, wherein the HA subtype-specific oligonucleotides detect and anneal to a HA gene segment, which amplifies a region comprising a diagnostically useful sequence.
7. The method according to claim 4, wherein the diagnostically useful sequence encompasses codons corresponding to amino acid residues at positions 226 and 228 of the HA protein, the identity of which are predictive for binding of influenza type A virus to receptors on mammalian cells.
8. The method according to claim 4, wherein the diagnostically useful sequence encompasses the host protease cleavage site within the HA gene, the sequence of which is predictive of the pathogenicity of influenza type A virus.
9. The method according to claim 2 or 5, wherein the NA subtype-specific oligonucleotides detect and anneal to a NA gene segment, which amplifies a region comprising a diagnostically useful sequence.
10. The method according to claim 9, wherein the diagnostically useful sequence encompasses one or more codons corresponding to amino acid residues between residues 820 to 880 of the NA protein, the identity of which is/are predictive of resistance of influenza type A virus to Oseltamivir (Tamiflu).
11. The method according to claim 2 or 5, wherein the PB2-specific oligonucleotides detect and anneal to a PB2 gene segment which amplifies a region comprising a diagnostically useful sequence.
12. The method according to claim 11, wherein the diagnostically useful sequence encompasses a codon corresponding to an amino acid residue at position 627 and/or 355 of the PB2 protein, the identity of which is predictive of replication efficiency of the influenza type A virus in mammals and ability to replicate in humans.
13. The method according to claim 5, wherein the HA subtype-specific oligonucleotides comprise sequences as set forth in two or more of SEQ ID NOs: 5, 6, 7, and SEQ ID NOs: 18, and 19.
14. The method according to claim 5, wherein the NA subtype-specific oligonucleotides comprise sequences as set forth in two or more of SEQ ID NOs: 9 to 17, and SEQ ID NOs: 20 to 22.
15. The method according to claim 5, wherein the PB2-specific oligonucleotides comprise sequences as set forth in two or more of SEQ ID NOs: 1, 2, 3 and 4.
16. The method of claim 1, wherein the analysis step comprises sequencing of the target sequence.
17. The method of claim 1, wherein the analysis step comprises restriction endonuclease digestion of the target sequence.
18. The method of claim 1, wherein the analysis step comprises hybridization of one or more molecular probes to the target sequence.
19. The method of claim 18, wherein the molecular probe is a labelled nucleic acid probe complementary to the entire sequence, or a section of the target sequence.
20. The method of claim 19, wherein the probe comprises a quantifiable marker.
21. The method of claim 1, wherein the analysis step comprises microarray or biochip analysis of the target sequence.
22. The method of claim 21, wherein a plurality of oligonucleotide probes complementary to one or more amplified gene segments or portions thereof are immobilised on a solid support and the amplified gene segments are incubated with the array of probes under conditions suitable to permit hybridization there between.
23. A method of diagnosing influenza type A infection in an individual, the method comprising: i. obtaining a biological sample, suspected of containing influenza virus particles, and optionally isolating genetic material from the sample; ii. amplifying a segment of at least one gene selected from HA, NA and PB2 using a set of oligonulceotides, wherein the segment of the at least one gene contains a target sequence that is predictive of pathogenicity, virulence or drug resistance; and iii. analysing the amplified target sequence.
24. The method according to claim 23, wherein the biological sample is selected from, but not restricted to blood, saliva, mucus, faeces, and/or bodily fluids.
25. The method according to claim 23, wherein the sample is from a mammal having at least one characteristic, with respect to influenza type A virus, including but not restricted to fever, coma, coughing, weakness, fatigue, aching, diarrhoea, nausea and vomiting.
26. The method according to claim 23, wherein the sample is from a mammal having no associated characteristics, with respect to influenza type A virus.
27. The method according to claim 23, wherein the sample is from an avian species.
28. The method according to claim 23, wherein the genetic material comprises RNA or cDNA.
29. The method according to claim 23, wherein the oligonucleotides are designed to detect multiple HA virus subtypes, multiple NA virus subtypes and/or multiple PB2 virus subtypes respectively.
30. The method according to claim 29, wherein the HA subtype-specific oligonucleotides comprise sequences as set forth in two or more of SEQ ID NOs: 5, 6, 7, and SEQ ID NOs: 18 and 19.
31. The method according to claim 29, wherein the NA subtype-specific oligonucleotides comprise sequences as set forth in two or more of SEQ ID NOs: 9 to 17, and SEQ ID NOs: 20 to 22.
32. The method according to claim 29, wherein the PB2-specific oligonucleotides comprise sequences as set forth in two or more of SEQ ID NOs: 1, 2, 3 and 4.
33. The method according to claim 23 wherein amplification is performed via Polymerase Chain Reaction (PCR).
34. The method according to claim 23, wherein the genetic material comprises RNA.
35. The method according to claim 34, wherein the RNA is amplified by Reverse Transcriptase-PCR to form cDNA.
36. The method according to claim 23, wherein amplification is performed via Real-Time PCR.
37. The method according to claim 23, wherein the analysis step comprises sequencing of the target sequence.
38. The method according to claim 23, wherein the analysis step comprises restriction endonuclease digestion of the target sequence.
39. The method according to claim 23, wherein the analysis step comprises hybridization of one or more molecular probes to the target sequence.
40. The method according to claim 39, wherein the molecular probe is a labelled nucleic acid probe complementary to the entire sequence, or a section of the target sequence.
41. The method according to claim 40, wherein the probe comprises a quantifiable marker.
42. The method according to claim 23, wherein the analysis step comprises microarray or biochip analysis of the target sequence.
43. The method according to claim 42, wherein a plurality of oligonucleotide probes complementary to one or more amplified gene segments or portions thereof are immobilised on a solid support and the amplified gene segments are incubated with the array of probes under conditions suitable to permit hybridization there between.
44. The method according to claim 23, wherein the sample is pretreated but does not require purification of the nucleic acids.
45. A test kit suitable for the detection of influenza type A from a biological sample, comprising: i. oligonucleotides for amplifying a segment of at least one gene selected from HA, NA and PB2, wherein the segment of the at least one gene contains a target sequence that is predictive of pathogenicity, virulence or drug resistance; ii. reagents for nucleic acid amplification, including at least one reagent selected from enzymes, pure water, sterile tubes, hot wax, filter tips, buffer and wash solution.
46. The test kit of claim 45, wherein the sample is pretreated but does not require purification of the nucleic acids.
47. The test kit of claim 45, wherein the biological sample is purified to contain a concentrated solution of DNA.
48. The test kit of claim 45, wherein the biological sample is purified to contain a concentrated solution of RNA.
49. The test kit of claim 45, further comprising at least one hybridisation probe able to indicate the presence of the target sequence.
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Publication number Priority date Publication date Assignee Title
CN108761077A (en) * 2018-08-06 2018-11-06 南京农业大学 Poultry diease viral disease protein chip antibody assay kit and its preparation method and application
CN109887547A (en) * 2019-03-06 2019-06-14 苏州浪潮智能科技有限公司 A kind of gene order compares filtering accelerated processing method, system and device
CN111575406A (en) * 2020-05-14 2020-08-25 张阳 Novel coronavirus rapid screening method and kit based on nucleic acid isothermal amplification

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Publication number Priority date Publication date Assignee Title
CN108761077A (en) * 2018-08-06 2018-11-06 南京农业大学 Poultry diease viral disease protein chip antibody assay kit and its preparation method and application
CN109887547A (en) * 2019-03-06 2019-06-14 苏州浪潮智能科技有限公司 A kind of gene order compares filtering accelerated processing method, system and device
CN111575406A (en) * 2020-05-14 2020-08-25 张阳 Novel coronavirus rapid screening method and kit based on nucleic acid isothermal amplification

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