(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`22 December 2005 (22.12.2005)
`
` (10) International Publication Number
`
`WO 2005/121367 A1
`
`(51) International Patent Classification7:
`C12N 15/11, 15/44
`
`C12Q 1/68,
`
`(21) International Application Number:
`PCT/SG2005/000187
`
`(22) International Filing Date:
`
`10 June 2005 (10.06.2005)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/578,353
`
`English
`
`English
`
`10 June 2004 (10.06.2004)
`
`US
`
`(71) Applicant (for all designated States except US): AGENCY
`FOR SCIENCE, TECHNOLOGY AND RESEARCH
`[SG/SG]; 20 Biopolis Way, #07—01 Centros, Singapore
`138668 (SG).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): REN, Ee Chee
`[MY/SG]; 62 Greenleaf View, Singapore 279301 (SG).
`NG, Lisa Fong Poh [SG/SG]; Blk 10B, #22—06 Braddell
`View, Singapore 579721 (SG). CI-HA, Jer lVIing [SG/SG];
`108 West Coast Rise, Singapore 127524 (SG).
`
`(74) Agent: YU, SARN, AUDREY & PARTNERS; 190 Mid—
`dle Road, #12—04, Singapore 188979 (SG).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI, NO, NZ,
`OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL,
`SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC,
`VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, MC, NL, PL, PT, RO,
`SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`* with international search report
`
`For twoiletter codes and other abbreviations, refer to the ”Guide
`ance Notes on Codes andAbbreviations” appearing at the begin—
`ning of each regular issue of the PCT Gazette.
`
`(54) Title: DIAGNOSTICS PRIMERS AND METHOD FOR DETECTING AVIAN INFLUENZA VIRUS SUBTYPE H5 AND
`H5N1
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`(57) Abstract: The present invention provides primers directed to conserved regions of the HA and NA genes of avian influenza
`virus subtypes H5 or H5Nl , and provides a method for detecting avian influenza subtype H5 or H5Nl .
`
`
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`WO 2005/121367
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`PCT/SG2005/000187
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`DIAGNOSTIC PRIMERS AND METHOD FOR DETECTING AVIAN
`
`INFLUENZA VIRUS SUBTYPE H5 AND H5N1
`
`CROSS-REFERENCE TO RELATED APPLICATION
`
`[0001]
`
`This application claims benefit and priority from US. provisional patent
`
`application No. 60/578,353, filed on June 10, 2004, the contents of which are
`
`incorporated herein by reference.
`
`FIELD OF THE INVENTION
`
`[0002]
`
`The present invention relates to a nucleic acid based detection method, more
`
`particularly, to primers and a method of detecting avian influenza virus.
`
`BACKGROUND OF THE INVENTION
`
`[0003]
`
`Three types of influenza viruses, types A, B, and C are known and they
`
`belong to a family of single—stranded negative-sense enveloped RNA Viruses called
`
`Orthomyxoviridae (Swayne, D. E., and D. L. Suarez (2000) Rev. Sci. Tech. 191463-482).
`
`The viral genome is approximately 12 000 to 15 000 nucleotides in length and comprises
`
`eight RNA segments (seven in Type C).
`
`[0004]
`
`Influenza A virus infects many animals such as humans, pigs, horses, marine
`
`mammals, and birds (Nicholson, K. G., et a1. (2005) Lancet 362:1733-1745). Its natural
`reservoir is in aquatic birds, and in avian species most influenza Virus infections cause
`
`mild localized infections of the respiratory and intestinal tract. However, the Virus can
`
`have high pathogenic effect in poultry, with sudden outbreaks causing high mortality
`
`rates in affected poultry populations. Highly pathogenic strains such as H5Nl cause
`
`system infections in which mmtality may reach 100% (Zeitlin, G. A., and M. J. Maslow
`
`(2005) Curr. Infect. Dis. Rep. 7 :193-199). In humans, influenza viruses cause a highly
`
`contagious acute respiratory disease that have resulted in epidemic and pandemic disease
`
`in humans (Cox, N. J., and K. Subbarao (1999) Lancet 354:1277-1282).
`
`[0005]
`
`Influenza A viruses can be classified into subtypes based on allelic variations
`
`in antigenic regions of two genes that encode surface glycoproteins, namely,
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`WO 2005/121367
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`PCT/SG2005/000187
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`hemagglutinin (HA) and neuraminidase (NA) which are required for viral attachment and
`
`cellular release. Other major Viral proteins include the nucleoprotein, the nucleocapsid
`
`structural protein, membrane proteins (M1 and M2), polymerases (PA, FBI and PB2),
`
`and non—structural proteins (NSI and N82).
`
`[0006]
`
`Currently, fifteen subtypes of HA (HI-H15) and nine NA (N 1-N9) antigenic
`
`variants are known in influenza A virus. Subtypes H5 and H7 can cause highly
`
`pathogenic infections in poultry and certain subtypes have been shown to cross the
`
`species barrier to humans. Previously, only three subtypes have been known to circulate
`
`in humans (H 1N1, H1N2, and H3N2). However, in recent years, the pathogenic H5Nl
`
`subtype of avian influenza A has been reported to cross the species barrier and infect
`
`humans as documented in Hong Kong in 1997 and 2003 (Peiris, J. S. M., et a1. (2004)
`
`Lancet 363:617-619; Yuen, K. Y., et al. (1998) Lancet 351: 467—471), leading to the
`
`death of some patients. Since late 2003, the H5N1 avian influenza A in poultry reached
`
`epidemic proportions with reports of serious outbreaks in several Asian countries
`
`including Vietnam, Thailand, South Korea, Laos, Cambodia, Indonesia, Japan and
`
`Malaysia (Centers for Disease Control and Prevention (CDC) (2004) Morb. Mortal.
`
`Wkly. Rep. 53:100-3; Hien T. T., et a1. (2004) N. Engl. J. Med. 350: 1179—1188) that
`
`resulted in massive culling of millions of poultry which had severe economic
`
`repercussions.
`
`[0007]
`
`In humans, the avian influenza Virus infects cells of the respiratory tract as
`
`well as the intestinal tract, liver, spleen, kidneys and other internal organs. Symptoms of
`
`avian flu infection include fever, respiratory difficulties including shortness of breath and
`
`cough, lymphopenia, diarrhea and difficulties regulating blood sugar levels. Due to the
`
`high pathogenicity of H5 subtypes, particularly H5N1, and their demonstrated ability to
`
`cross over to infect humans, there is a significant economic and public health risk
`
`associated with these viral strains, including a real epidemic and pandemic threat.
`
`[0008]
`
`As a result, H5N1 avian influenza A Virus represents a potential danger to
`
`human health not only in Asia but to the world. In addition to containment procedures,
`
`sensitive detection assays for early diagnosis are vital to lower the chances of spread and
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`reduce the risk of development into an epidemic.
`
`[0009]
`
`Currently, there are a variety of techniques that can be used to detect H5 and
`
`H5N1 subtypes of avian influenza Virus in biological samples, including nucleic acid
`
`sequence—based amplification (NASBA) methods that amplify RNA, viral cultures,
`
`reverse—transcription polymerase chain reaction (RT—PCR) methods that amplify DNA
`
`transcribed from the Viral RNA genome, hemagglutination inhibition and various
`
`fluorescence and enzyme—linked irnmunoassays (ELISAS).
`
`[0010]
`
`In particular, PCT publication WO 02/29118 by So et a1. describes a NASBA
`
`assay and kit for detecting H5 subtypes of avian flu Virus. Hien et al. (2004, New Eng. J
`
`ofMed. 350(12):1179-1188) describe the use of antigen tests using various fluorescence
`
`and enzyme-linked immunoassays. Lau et al. (2003, Biochem. Biophys. Res. Comm.
`
`313:336—342) describes a NASBA method for detection of H5 or H7 subtypes of avian
`
`influenza Virus. Lee et al. (2001, J. Virol. Methods 97: 13-32) and Payungporn et al.
`
`(2004, Viral Immunol. 17:588-593) describe RT-PCR assays for identification and
`
`subtyping or detection of avian flu virus subtypes. However, each of these methods uses
`
`genetic information derived from only a few isolates or variants of H5 or H5N1 to
`
`confirm the presence of virus. Furthermore, these assays are reported to be low in
`
`specificity and sensitivity. Clinically, the low sensitivity of these diagnostics may limit
`
`the usefulness for reliable detection of influenza A (H5N1) virus in humans. Therefore,
`
`there is an urgent need for sensitive diagnostic tests useful for rapid and early diagnosis.
`
`SUMMARY OF THE INVENTION
`
`[0011]
`
`Based on sequence comparison of the HA gene from greater than 200 H5
`
`isolates and greater than 100 H5N1 isolates, and on sequence comparison of the NA gene
`
`from approximately 70 HSNl isolates, a series of primers directed to conserved regions
`
`Within these genes has been developed. These primers are useful to screen for a wide
`
`variety of H5 and H5N1 isolates, and allow for detection methods that are rapid, specific
`
`and sensitive.
`
`[0012]
`
`Thus, in one aspect, the present invention provides a primer comprising a
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`sequence of any one of SEQ ID N021 to SEQ ID NO:114. In another aspect, there is
`
`provided a primer comprising a target annealing sequence and a non-influenza A virus
`
`sequence, wherein the target annealing sequence comprises a sequence of any one of
`
`SEQ ID NO:1 to SEQ ID NO:114.
`
`[0013]
`
`These primers are useful for detecting the presence of avian influenza Virus
`
`H5 or H5N1 in a sample, for example a sample derived from an organism suspected of
`
`carrying such a virus, and may be used in a reverse—transcription polymerase chain
`
`reaction in order to detect the presence of Virus in the sample.
`
`[001 4]
`
`Thus, in another aspect the present invention provides a method for detecting
`
`influenza A virus subtype H5 or HSNl in a sample comprising amplifying DNA reverse
`
`transcribed from RNA obtained from the sample using one or more primers each
`
`comprising a sequence of any one of SEQ ID NO:1 to SEQ ID NO:114; and detecting a
`
`product of amplification, wherein the presence of the product of amplification indicates
`
`the presence of an avian influenza virus subtype H5 or H5N1 in the sample.
`
`[001 5]
`
`The methods described herein can be used to detect a wide variety of H5 and
`
`H5N1 influenza A virus isolates. Using a one-step method, in which RNA is reverse-
`
`transcribed and product is amplified in a single reaction tube, allows for a reduction in
`
`detection time, minimizes sample manipulation and lowers the risk of cross-
`
`contamination of samples. Thus, the described methods using the described primers may
`
`be useful for early detection and/or diagnosis of H5 and H5N1 influenza A infection.
`
`Furthermore, these methods can be used to determine approximate viral load in a sample,
`
`which application is useful in clinical and public health management settings.
`
`[0016]
`
`The primers of the invention may be useful in other amplification methods,
`
`such as nucleic acid based sequence amplification methods to detect the presence of
`
`avian influenza virus subtype H5 or H5N1 in a sample. The primers of the invention may
`
`also be usefiil for sequencing DNA corresponding to the HA or NA gene of avian
`
`influenza virus subtype H5 or H5N1.
`
`[001 7]
`
`In another aspect, there is provided a method of detecting influenza A Virus
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`subtype H5 or H5N1 in a sample comprising contacting the sample with a primer
`
`immobilized on a support, said primer comprising a sequence of any one of SEQ ID
`
`NO:1 to SEQ ID NO:114, under conditions suitable for hybridizing the primer and the
`
`sample; and detecting hybridization of the immobilized primer and the sample.
`
`[001 8]
`
`In a flirther aspect, there is provided a method of detecting influenza A virus
`
`subtype H5 or H5N1 in a sample comprising contacting the sample with a nucleic acid
`
`microarray, the nucleic acid microarray comprising one or more primers, each of said
`
`primers comprising a sequence of any one of SEQ ID NO:1 to SEQ ID NO:114, under
`
`conditions suitable for hybridizing the one or more primers and the sample; and detecting
`
`hybridization of the one or more primers and the sample.
`
`[001 9]
`
`In another aspect, there is provided a nucleic acid microarray comprising a
`
`primer, said primer comprising a sequence of any one of SEQ ID NO:1 to SEQ ID
`
`NO: 1 14.
`
`[0020]
`
`In a further aspect, there is provided a kit comprising a primer as defined
`
`herein and instructions for detecting influenza A virus subtype H5 or H5N1 in a sample.
`
`[0021]
`
`Other aspects and features of the present invention will become apparent to
`
`those of ordinary skill in the art upon review of the following description of specific
`
`embodiments of the invention in conjunction with the accompanying figures.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0022]
`
`Figure 1 is a schematic diagram representing the HA gene, and depicting the
`
`location of exemplary forward and reverse primers of the present invention (beginning
`
`with “gisAF”) and of primers known in the art (beginning with “TW”, “VM” or “HK”);
`
`[0023]
`
`Figure 2 is a photograph of an agarose gel displaying PCR amplification
`
`products prepared by a gel-based PCR approach using exemplary primers (sets 1 to 8) of
`
`the invention to amplify template DNA reverse transcribed from RNA of an H5N1
`
`isolate;
`
`[0024]
`
`Figure 3 is a photograph of an agarose gel displaying the relative amounts of
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`amplification product obtained using varying amounts of template and primer set 3 used
`
`in Figure 2;
`
`[0025]
`
`Figure 4 is a photograph of an agarose gel displaying the relative amounts of
`
`amplification product obtained using varying amounts of template and primer set 5 used
`
`in Figure 2;
`
`[0026]
`
`Figure 5 is a photograph of an agarose gel displaying the relative amounts of
`
`amplification product obtained using varying amounts of template and primer sets 8
`
`(upper bands) and 6 (lower bands) used in Figure 2;
`
`[0027]
`
`Figure 6 is a photograph of an agarose gel displaying PCR amplification
`
`products prepared by a real time PCR approach with SYBR green dye, using exemplary
`
`primers (sets 1 to 8) of the invention to amplify template DNA reverse transcribed from
`
`RNA of an H5N1 isolate;
`
`[0028]
`
`Figure 7 is an amplification curve obtained during the real time PCR
`
`amplification reaction using primer set 1 of Figure 6;
`
`[0029]
`
`Figure 8 is an amplification curve obtained during the real time PCR
`
`amplification reaction using primer set 2 of Figure 6;
`
`[0030]
`
`Figure 9 is an amplification curve obtained during the real time PCR
`
`amplification reaction using primer set 3 of Figure 6;
`
`[0031]
`
`Figure 10 is an amplification curve obtained during the real time PCR
`
`amplification reaction using primer set 4 of Figure 6;
`
`[0032]
`
`Figure 11 is an amplification curve obtained during the real time PCR
`
`amplification reaction using primer set 5 of Figure 6;
`
`[0033]
`
`Figure 12 is an amplification curve obtained during the real time PCR
`
`amplification reaction using primer set 6 of Figure 6;
`
`[0034]
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`Figure 13 is an amplification curve obtained during the real time PCR
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`amplification reaction using primer set 7 of Figure 6;
`
`[0035]
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`Figure 14 is an amplification curve obtained during the real time PCR
`
`amplification reaction using primer set 8 of Figure 6;
`
`[0036]
`
`Figure 15 is a melting curve obtained at the end of the real time PCR
`
`amplification reaction using primer sets 1 and 2 of Figure 6;
`
`[0037]
`
`Figure 16 is a melting curve obtained at the end of the real time PCR
`
`amplification reaction using primer sets 3, 4 and 5 of Figure 6;
`
`[0038]
`
`Figure 17 is a melting curve obtained at the end of the real time PCR
`
`amplification reaction using primer sets 5 and 6 of Figure 6;
`
`[0039]
`
`Figure 18 is a melting curve obtained at the end of the real time PCR
`
`amplification reaction using primer sets 7 and 8 of Figure 6;
`
`[0040]
`
`Figures 19 A and B are photographs of agarose gels demonstrating the
`
`detection of H5N1 avian influenza A Virus by one-step RT-PCR; A: amplification of
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`serially diluted in vitro—transcribed single-stranded RNA; B: Specific detection of H5N1
`
`avian influenza virus from field samples;
`
`[0041]
`
`Figures 20 A and B are photographs of agarose gels of PCR products
`
`obtained using either A: a two-step RT—PCR reaction; or B: a one—step RT—PCR reaction;
`
`[0042]
`
`Figure 20C depicts the results of real time PCR using primer set 6;
`
`[0043]
`
`Figures 21 A, B and C are photographs of agarose gels demonstrating the use
`
`of exemplary primers of the invention on field samples to detect H5N1 avian influenza
`
`virus; A: samples of allantoic fluid; B: samples of homogenized tissues; and C:
`
`comparison of an in-house H5 primer set with an H5Nl primer set;
`
`[0044]
`
`Figures 22 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 9) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
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`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0045]
`
`Figures 23 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 10) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0046]
`
`Figures 24 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 11) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0047]
`
`Figures 25 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 12) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0048]
`
`Figures 26 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 13) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0049]
`
`Figures 27 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 14) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0050]
`
`Figures 28 A, B and C depict the results of real time PCR with SYBR green
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`dye using exemplary primers (set 15) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0051]
`
`Figures 29 A, B and C depict the results of real time PCR With SYBR green
`
`dye using exemplary primers (set 16) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0052]
`
`Figures 30 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 17) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0053]
`
`Figures 31 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 1 8) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0054]
`
`Figures 32 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 19) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
`
`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0055]
`
`Figures 33 A, B and C depict the results of real time PCR with SYBR green
`
`dye using exemplary primers (set 20) directed against the NA gene of H5N1 influenza A;
`
`A is an amplification curve obtained during the real time PCR amplification reaction; B
`
`are melting curves obtained at the end of the amplification reaction and C is a photograph
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`of a 1.5% agarose gel displaying the PCR amplification products;
`
`[0056]
`
`Figures 34 A B, C and D depict the results of real time PCR with SYBR
`
`green dye using exemplary primers (set 21) directed against the HA gene of H5N1
`
`influenza A; A is an amplification curve obtained during the real time PCR amplification
`
`reaction; B is an RNA standard curve; C are melting curves obtained at the end of the
`
`amplification reaction and D is a photograph of a 1.5% agarose gel displaying the PCR
`
`amplification products;
`
`[0057]
`
`Figures 35 A B, C and D depict the results of real time PCR with SYBR
`
`green dye using exemplary primers (set 22) directed against the HA gene of H5N1
`
`influenza A; A is an amplification curve obtained during the real time PCR amplification
`
`reaction; B is an RNA standard curve; C are melting curves obtained at the end of the
`
`amplification reaction and D is a photograph of a 1.5% agarose gel displaying the PCR
`
`amplification products;
`
`[0058]
`
`Figures 36 A, B, C and D depict the results of real time PCR with SYBR
`
`green dye using exemplary primers (set 23) directed against the HA gene of H5 influenza
`
`A (H5N1 (QS 1 to QS 5), H5N2 (a) and H5N3 (b)); A is an amplification curve obtained
`
`during the real time PCR amplification reaction; B is an RNA standard curve;C are
`
`melting curves obtained at the end of the amplification reaction and D is a photograph of
`
`a 1.5% agarose gel displaying the PCR amplification products;
`
`[0059]
`
`Figure 37 is a photograph of an agarose gel displaying the relative amomts of
`
`amplification product obtained by a one-step RT—PCR method, using varying amounts of
`
`template and primer set 10;
`
`[0060]
`
`Figure 38 is a photograph of an agarose gel displaying the relative amounts of
`
`amplification product obtained by a one—step RT—PCR method, using varying amounts of
`
`template and primer set 11;
`
`[0061]
`
`Figure 39 is a photograph of an agarose gel displaying the relative amounts of
`
`amplification product obtained by a one-step RT-PCR method, using varying amounts of
`
`template and primer set 13;
`
`10
`
`

`

`WO 2005/121367
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`PCT/SG2005/000187
`
`[0062]
`
`Figure 40 is a photograph of an agarose gel displaying the relative amounts of
`
`amplification product obtained by a one—step RT-PCR method, using varying amounts of
`
`template and primer set 16;
`
`[0063]
`
`Figure 41 is a photograph of an agarose gel displaying the relative amounts of
`
`amplification product obtained by a two—step RT-PCR method, using varying amounts of
`
`template and primer set 12 ;
`
`[0064]
`
`Figure 42 is a photograph of an agarose gel displaying the relative amounts of
`
`amplification product obtained by a two—step RT-PCR method, using varying amounts of
`
`template and primer set 15 ;
`
`[0065]
`
`Figures 43 A and B are photographs of agarose gels displaying the relative
`
`amounts of amplification product using varying amounts of template and primer set 23,
`
`obtained by A: a one-step RT—PCR method; and B: a two-step RT-PCR method;
`
`[0066]
`
`Figures 44 is an amplification curve obtained using the TaqmanTM real time
`
`PCR method and primer set 24, directed against the HA gene of subtype H5;
`
`[0067]
`
`Figures 45 is an amplification curve obtained using the TaqmanTM real time
`
`PCR method and primer set 25, directed against the HA gene of subtype HSNl ; and
`
`[0068]
`
`Figures 46 is an amplification curve obtained using the TaqmanTM real time
`
`PCR method and primer set 26, directed against the HA gene of subtype H5Nl .
`
`DETAILED DESCRIPTION
`
`[0069]
`
`RNA viruses, including the influenza A virus, tend to have high mutation
`
`rates due to the low fidelity nature of RNA replication when compared to DNA
`
`replication. As a result, influenza viruses tend to evolve rapidly. Furthermore, influenza
`
`A viruses tend to undergo genetic reassortment between viral strains, which mechanism
`
`has contributed to the development of the various HA and NA subtypes. The inventors
`
`compared the sequence of the hemagglutinin (“HA”) gene from more than 200 influenza
`
`A H5 isolates, and more than 100 influenza A H5N1 isolates. As well, the inventors
`
`compared the sequence of the neuraminidase (“NA”) gene from approximately 70
`
`ll
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`WO 2005/121367
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`PCT/SG2005/000187
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`influenza A H5N1 isolates. Surprisingly, despite the high mutation rate within influenza
`
`viruses, the inventors have discovered short regions of highly conserved sequences
`
`unique to specific subtypes, which regions are suitable to identify or detect the presence
`
`of those subtypes in a sample.
`
`[0070]
`
`The sequences used in the comparison were obtained from publicly available
`
`databases and were compared using a variety of sequence comparison software, including
`
`the software ClustalW.
`
`[0071]
`
`These sequence comparisons allowed the inventors to develop forward and
`
`reverse primers set out in SEQ ID NO:1 to SEQ ID N011 14, directed to conserved
`
`regions of the HA gene or the NA gene of avian influenza virus subtypes H5 or H5N1,
`
`for use in a detection assay, for example, reverse—transcription followed by polymerase
`
`chain reaction amplification (“RT-PCR”). The comparison of such a large number of
`
`viral isolates allowed for the design of primers directed to well-conserved regions of the
`
`HA or NA gene, thus targeting regions that are less likely to be affected by mutational
`
`changes and thereby providing primers that can detect a larger pool of H5 or H5Nl
`
`variants than primers that are currently available.
`
`[0072]
`
`The term “isolate” as used herein refers to a particular virus or clonal
`
`population of virus particles, isolated from a particular biological source, such as a
`
`patient, which has a particular genetic sequence. Different isolates may vary at only one
`
`or several nucleotides, and may still fall Within the same viral subtype. A viral subtype
`
`refers to any of the subtypes of HA or subtypes of NA classified according to the
`
`antigenicity of these glycoproteins.
`
`[0073]
`
`The inventors found that in certain conserved regions, one or more nucleotides
`
`at a specific location varied between isolates. For those regions, a family of primers has
`
`been developed, each primer within the family being based on a conserved sequence of
`
`the HA or the NA gene, but varying at one or more particular bases within the conserved
`
`sequence.
`
`[0074]
`
`Thus, in one aspect the invention provides a primer comprising a sequence as
`
`12
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`WO 2005/121367
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`PCT/SG2005/000187
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`set out in any one of SEQ ID N021 to SEQ ID N02114.
`
`[0075]
`
`As will be understood by a skilled person, a “primer” is a single—stranded
`
`DNA or RNA molecule of defined sequence that can base pair to a second DNA or RNA
`
`molecule that contains a complementary sequence (the target). The stability of the
`
`resulting hybrid molecule depends upon the extent of the base pairing that occurs, and is
`
`affected by parameters such as the degree of complementarity between the primer and
`
`target molecule and the degree of stringency of the hybridization conditions. The degree
`
`of hybridization stringency is affected by parameters such as the temperature, salt
`
`concentration, and concentration of organic molecules, such as formamide, and may be
`
`determined using methods that are known to those skilled in the art. Primers can be used
`
`for methods involving nucleic acid hybridization, such as nucleic acid sequencing,
`
`nucleic acid amplification by the polymerase chain reaction, single stranded
`
`conformational polymorphism (SSCP) analysis, restriction fragment polymorphism
`
`(RFLP) analysis, Southern hybridization, northern hybridization, in situ hybridization,
`
`electrophoretic mobility shift assay (EMSA), nucleic acid microarrays, and other methods
`
`that are known to those skilled in the art.
`
`[0076]
`
`The term “RNA” refers to a sequence of two or more covalently bonded,
`
`naturally occurring or modified ribonucleotides. The RNA may be single stranded or
`
`double stranded. The term “DNA” refers to a sequence of two or more covalently
`
`bonded, naturally occurring or modified deoxyribonucleotides, including cDNA and
`
`synthetic (e.g., chemically synthesized) DNA, and may be double stranded or single
`
`stranded. By “reverse transcribed DNA” or “DNA reverse transcribed from” is meant
`
`complementary or copy DNA (cDNA) produced from an RNA template by the action of
`
`RNA—dependent DNA polymerase (reverse transcriptase).
`
`[0077]
`
`Avian influenza virus is a single stranded RNA virus and in some
`
`embodiments, the primer has a DNA sequence that corresponds to the RNA sequence of
`
`a conserved region of the HA gene of avian influenza virus subtype H5 or H5N1 (SEQ
`
`ID N021 to SEQ ID N031 and SEQ ID N02112), as set out in Table 1. Such primers
`
`may be used as a forward primer when sequencing or amplifying DNA reverse
`
`13
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`transcribed from the HA gene of subtypes H5 or
`
`H5N1.
`
`e H5 or H5N1
`Table 1:
`F
`orward Primers for the HA Gene of Sub
`
`Start
`
`
`
`
`Nucleotlde
`Sequence (5’ to 3’)
`
`
`84
`ATT TGC ATT GGT TAC CAT GCA AA
`gisAFHSHlaF —
`
`ATT TGC ATT GGT TAT CAT GCA AA
`
`gisAFHSHle
`84
`
`
`ATC TGC ATT GGT TAT CAT GCA AA
`84
`gisAFHSchF
`
`84
`ATC TGC ATC GGT TAT CAT GCA AA
`
`
`
`
`
`
`
`
`
`
`SEQ ID
`NO:
`
`1 2 3 4 5
`
`
`
`
`
`
`
`
`gisAFHSHldF
`
`gisAFHSHleF
`
`gisAFH5H1 fF
`
`gisAFH5H3aF
`
`g1sAFH5H3bF
`
`84
`
`84
`
`248
`
`248
`
`ATT TGC ATC GGT TAC CAT GCA AA
`
`ATT TGC ATT GGT CAT CAT GCA AA
`
`GCT GGA TGG CTC CTC GGA AAC CC
`
`GCT GGA TGG CTC CTT GGA AAT CC
`
`g1sAFH5H3cF
`
`
`248
`GCT GGA TGG CTC CTC GGA AAT CC
`
`248
`
`
`
`
`
`
`GCC GGA TGG CTT TTG GGG AAT CC
`
`GCT GGG TGG CTT CTT GGA AAC CC
`
`
`
`
`GCT GGG TGG CTT CTT GGA AAT CC
`
`
`GCT GGA TGG CTT CTC GGA AAT CC
`
`GCT GGG TGG CTC CTC GGA AAC CC
`
`CAG ATT TGC ATT GGT TAC CAT GC
`
`
`
`13
`gisAFH5H3hF
`14
`15
`
`gisAFH5H3iF
`
`
`
`7
`
`10
`
`11
`
`12
`
`16
`
`17
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`29
`
`800
`
` gisAFH5H3jF
`
`
`
`
`
`gisAFH5H3kF
`
`gisAFH5H31F
`
`gisAFHSNlHlF
`
`gisAFH5N1H2aF
`
`glsAFHSNlHZbF
`
`gisAFHSNlHZcF
`
`GTT GAC ACA ATA ATG GAA AAG
`
`
`AA
`
`GTT GAT ACA ATA ATG GAA AAG AA
`
`
`GTT GAC ACA ATA ATG GAG AAG
`AA
`
`GCT GGA TGG CTC CTC GGA AAC CC
`gisAFH5N1H3F
`248
`
`248
`
`12
`
`2
`
`22
`
`1
`122
`
`gisAFHSNlHGaF
`
`gisAFH5N1H6bF
`
`gisAFH5N1H7aF
`
`366
`366
`
`gisAFH5N1H7bF
`
`800
`
`gisAFH5N1H9F
`
`
`
`gisAFH5N1H12aF
`
`