WO2011136743A1 - Méthodes et/ou amorces de détection de la grippe a - Google Patents

Méthodes et/ou amorces de détection de la grippe a Download PDF

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Publication number
WO2011136743A1
WO2011136743A1 PCT/SG2011/000164 SG2011000164W WO2011136743A1 WO 2011136743 A1 WO2011136743 A1 WO 2011136743A1 SG 2011000164 W SG2011000164 W SG 2011000164W WO 2011136743 A1 WO2011136743 A1 WO 2011136743A1
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WO
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Prior art keywords
seq
fragment
derivative
mutation
influenza
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PCT/SG2011/000164
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English (en)
Inventor
Charlie Lee Wah Heng
Ken Sung Wing Kin
Yang Sun Chan
Christopher Wong Wing Cheong
Martin Lloyd Hibberd
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Agency For Science, Technology And Research
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Publication of WO2011136743A1 publication Critical patent/WO2011136743A1/fr

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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
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to methods and reagents for detecting Influenza A virus.
  • the invention relates to the detection of influenza A virus, subtype Pandemic H1 N1 (2009).
  • Influenza A is an infectious disease of animals caused by type A strains of the influenza virus that normally infect animals, including birds, pigs and humans. There are many subtypes of the influenza A virus. These subtypes are based on haemagglutinin (HA) segment 4, which has 14 varieties and neuraminidase (NA) segment 6, which has 9 varieties. It is these two segments of the virus that cause virulence.
  • HA haemagglutinin
  • NA neuraminidase
  • influenza A subtype, H1 N1 is known to mutate quickly, and has been shown to be highly pathogenic and can cause severe diseases in man.
  • H1 1 has been viewed as a model for rapid genetic evolution and pandemic change.
  • evolution is explained to be the selection of frequent copy errors generated by a polymerase complex that lacks a copy function. These errors are then selected for an evolutionary advantage, such as evasion of the immune response of the host, which allows the influenza to expand and fix the selected mutation.
  • influenza viruses employ recombination for rapid evolution via homologous recombination. Therefore, mutation in influenza occurs frequently and these variants may include properties such as anti-viral drug resistance, ability to escape from immune recognition and the like.
  • H1 N1 Influenza A 2009 which was first detected in Singapore in May 2009 and has been shown to be highly virulent, particularly in man.
  • WO/2009/144667, WO/2017116270 and the Recommended laboratory tests to identify avian influenza A virus in specimens from human provide different PCR primers for detecting influenza A in general.
  • many of these primers may not be very sensitive and/or specific to the pandemic H1 N1(2009) and their mutated forms.
  • the present invention addresses the problems above, and in particular provides highly sensitive and specific oligonucleotides, fragment(s) and/or derivative(s) thereof useful in a method of detecting Influenza A virus in patient specimens.
  • the invention relates to the detection of influenza A virus, subtype pandemic H1 N1(2009).
  • the primers of the present invention may be more sensitive, specific and may be able to obtain high yield of amplicon by single gene amplification.
  • the increased sensitivity of the primers means that the risk of a false negative result may be reduced and the primers may be able to detect virus even in patients with low virus titres.
  • the primers of the present invention thus may provide rapid and cost-effective diagnostic and prognostic reagents for determining infection by influenza A viruses, in particular subtype H1 N1 (2009) viruses and/or disease conditions associated therewith-. These primers may provide an informative influenza assay that can be performed in the field, i.e., at the point of care ("POC").
  • the present invention provides at least one isolated oligonucleotide comprising, consisting essentially of, or consisting of at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:1 to SEQ ID NO:9, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof.
  • the oligonucleotide may be capable of binding to and/or being amplified from influenza A virus.
  • the influenza A virus may be of subtype pandemic H1 N1 (2009).
  • the present invention provides at least one pair of oligonucleotides comprising, consisting essentially of, or consisting of at least one forward primer and at least one reverse primer, wherein the forward primer comprises, consists essentially of or consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:1 , SEQ ID NO:4 and SEQ ID NO:7, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof and the reverse primer comprises, consists essentially of or consists of at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof.
  • the present invention provides a set of oligonucleotides comprising, consisting essentially of or consisting of a pair of oligonucleotides according to any aspect of the present invention and at least one probe, the probe comprising, consisting essentially of or consisting of at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof.
  • the present invention provides a set of oligonucleotides comprising, consisting essentially of or consisting of:
  • a forward primer comprising, consisting essentially of or consisting of SEQ ID NO:1 , fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof
  • a reverse primer comprising, consisting essentially of or consisting of SEQ ID NO:2, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof and a probe comprising, consisting essentially of or consisting of SEQ ID NO:3, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof;
  • a forward primer comprising, consisting essentially of or consisting of SEQ ID NO:4, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof
  • a reverse primer comprising, consisting essentially of or consisting of
  • SEQ ID NO:5 fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof and a probe comprising, consisting essentially of or consisting of SEQ ID NO:6, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof; and/or
  • a forward primer comprising, consisting essentially of or consisting of SEQ ID NO:7, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof
  • a reverse primer comprising, consisting essentially of or consisting of SEQ ID NO:8, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof
  • a probe comprising, consisting essentially of or consisting of SEQ ID NO:9, fragment(s), derivative(s), mutation(s), and complementary sequence(s) thereof.
  • the present invention provides an amplicon amplified from Influenza A using at least one pair of oligonucleotides according to any aspect of the present invention.
  • the present invention provides at least one method of detecting the presence of Influenza A virus in a biological sample, the method comprising the steps of:
  • step (c) detecting any binding resulting from the contacting in step (b) whereby the influenza A virus is present when binding is detected.
  • the influenza A virus may be of subtype pandemic H1N1(2009).
  • the present invention provides at least one method of amplifying nucleic acid of influenza A virus of subtype H1 N1(2009), wherein said method comprises carrying out a polymerase chain reaction using at least one forward primer according to any aspect of the present invention and at least one reverse primer according to any aspect of the present invention.
  • the present invention provides a kit for the detection of influenza A virus of subtype H1 N1(2009), the kit comprising at least one oligonucleotide, a pair of oligonucleotides or a set of oligonucleotides according to any aspect of the present invention and/or a sequencing primer according to any aspect of the present invention. All primers and probes may be mixed to construct multiplex one- step fluorescence probe-based real-time PCR in one-tube with high specificity and high sensitivity.
  • the oligonucleotides of the present invention may be capable of binding to and/or being amplified from Influenza A virus.
  • the Influenza A virus may be of subtype H1 N1.
  • the primers may be specific to segment 4 of the Influenza A virus of subtype Pandemic H1 N1 (2009).
  • Examples of the genomes of Influenza A virus of subtype Pandemic H1 N1 includes but is not limited to the list online in www.ncbi.nlm.nih.gov/genomes/FLU/SwineFlu.html.
  • the primers of the present application may target any genome of Influenza A virus of subtype Pandemic H1 N1 (2009).
  • biological sample is herein defined as a sample of any tissue and/or fluid from at least one animal and/or plant.
  • Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste.
  • Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.
  • Environmental samples include environmental material such as surface matter, soil, water, air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the methods disclosed herein.
  • a biological sample may be of any tissue and/or fluid from at least a human being.
  • complementary is used herein in reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence "5'-A-G-T-3',” is complementary to the sequence "3'-T-C-A-5 ⁇ "
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
  • the "complementary sequence” refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "anti-parallel association.”
  • Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids disclosed herein and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
  • oligonucleotide is complementary to a region of a target nucleic acid and a second oligonucleotide has complementary to the same region (or a portion of this region) a "region of overlap" exists along the target nucleic acid.
  • the degree of overlap may vary depending upon the extent of the complementarity.
  • comprising is herein defined as "including principally, but not necessarily solely”.
  • the term “comprising” will be automatically read by the person skilled in the art as including “consisting of.
  • the variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly varied meanings.
  • the term “derivative,” is herein defined as the chemical modification of the oligonucleotides of the present invention, or of a polynucleotide sequence complementary to the oligonucleotides. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • fragment is herein defined as an incomplete or isolated portion of the full sequence of an oligonucleotide which comprises the active/binding site(s) that confers the sequence with the characteristics and function of the oligonucleotide. In particular, it may be shorter by at least one nucleotide or amino acid. More in particular, the fragment comprises the binding site(s) that enable the oligonucleotide to bind to influenza A virus, particularly Influenza A virus of subtype Pandemic H1 N1 (2009).
  • a fragment of the oligonucleotides of the present invention may be about 20 nucleotides in length. In particular, the length of the fragment may be at least about 10 nucleotides in length.
  • the fragment of the forward primer may comprise at least 10, 12, 15, 18, 19, 20 or 22 consecutive nucleotides of SEQ ID NO:1
  • the reverse primer may comprise at least 10, 13, 15, 18, 19, 20, 21 or 22, consecutive nucleotides of SEQ ID NO:2
  • the fragment of the primer may be at least 15 nucleotides in length.
  • IC internal control
  • the term "internal control (IC) molecule” is herein defined as the in vitro transcribed oligonucleotide molecule which is co-amplified by the same primer set for influenza virus used in the method of the present invention.
  • the IC may be mixed in the reaction mixture to monitor the performance of PC to avoid false negative results.
  • the probe to detect this IC molecule may be specific to the interior part of this molecule. This interior part may be artificially designed and may not occur in nature.
  • influenza virus as used in the context of the invention includes all subtypes of influenza viruses that fall under the categories of "avian influenza viruses” and "human influenza viruses".
  • the influenza viruses may be influenza A, B or C viruses.
  • influenza A virus may include but are not limited to H1 1 , H3N2, H5N1 , H5N2, H5N8, H5N9, H7N2, H7N3, H7N4, H7N7, H9N2, Pandemic H1 N1 (2009) and the like.
  • mutation is herein defined as a change in the nucleic acid sequence of a length of nucleotides.
  • a person skilled in the art will appreciate that small mutations, particularly point mutations of substitution, deletion and/or insertion has little impact on the stretch of nucleotides, particularly when the nucleic acids are used as probes. Accordingly, the oligonucleotide(s) according to the present invention encompasses mutation(s) of substitution(s), deletion(s) and/or insertion(s) of at least one nucleotide.
  • oligonucleotide(s) and derivative(s) thereof may also function as probe(s) and hence, any oligonucleotide(s) referred to herein also encompasses their mutations and derivatives. For example, if mutations occur at a few base positions at any primer hybridization site of the target gene, particularly to the 5'-terminal, the sequence of primers may not affect the sensitivity and the specificity of the primers.
  • nucleic acid in the biological sample refers to any sample that contains nucleic acids (RNA or DNA).
  • sources of nucleic acids are biological samples including, but not limited to blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.
  • the oligonucleotides according to the present invention may be useful as primers and/or probes and may be used in methods for specificall detecting influenza A, particularly, pandemic H1 N1 (2009), in a sample containing either one or more strains of influenza and/or other unrelated viruses/microscopic organisms.
  • These nucleotide sequences of the primers and probes of the present invention are designed to hybridize specifically to regions of the influenza A genomes that are unique to the genome of subtype pandemic H1 1 (2009), that are also conserved across many viruses within each strain.
  • the primers according to any aspect of the present invention may also be used to distinguish the genotype of influenza A of subtype pandemic H1 N1 (2009) from other subtypes of influenza A.
  • the oligonucleotide sequence may be between 13 and 35 linked nucleotides in length and may comprise at least 70% sequence identity to any of the sequences selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:9.
  • a skilled person will appreciate that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction.
  • a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event, (e.g., for example, a loop structure or a hairpin structure).
  • sequence of the oligonucleotide may have 80%, 85%, 90%, 95% or 98% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:9.
  • Percent homology, sequence identity or complementarity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wl), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981 , 2, 482-489).
  • Gap program Widesin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wl
  • a skilled person is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product.
  • a set of oligonucleotides may comprise a pair of oligonucleotides according to any aspect of the present invention and at least one probe.
  • the probe may be labeled with a fluorescent dye at 5' and 3' ends thereof.
  • the 5'-labeled fluorescent dye may include, but are not limited to, 6-carboxyfluorescein (FAM), hexachloro-6- carboxyfluorescein (HEX), tetrachloro-o-carboxyfluorescein, and Cyanine-5 (Cy5).
  • Examples of the 3'-labeled fluorescent dye may include, but are not limited to, 5- carboxytetramethylrhodamine (TAMRA) and black hole quencher- 1 ,2,3 (BHQ-1 ,2,3);
  • the oligonucleotides of the present invention may be used in various nucleic acid amplification techniques known in the art, such as, for example, Polymerase Chain Reaction (PCR), Nucleic Acid Sequence Based Amplification (NASBA), Transcription- Mediated Amplification (TMA), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), thermophilic SDA (tSDA) or Ligation-Mediated Amplification (LMA).
  • PCR Polymerase Chain Reaction
  • NASBA Nucleic Acid Sequence Based Amplification
  • TMA Transcription- Mediated Amplification
  • RCA Rolling Circle Amplification
  • SDA Strand Displacement Amplification
  • tSDA thermophilic SDA
  • LMA Ligation-Mediated Amplification
  • the oligonucleotides of the present invention may also be used in a variety of methods known to one of ordinary skill in the art for direct detection of influenza A without amplification through direct hybridization with viral nucleic acids, or to detect DNA or RNA copies of viral nu
  • the oligonucleotide according to any aspect of the present invention may be used in a method for the detection of influenza from either a clinical or a culture sample, wherein the clinical samples may include but are not limited to, nasopharyngeal, nasal and throat swabs as well as nasopharyngeal aspirates and washes.
  • the clinical sample may undergo preliminary processing prior to testing to allow more efficient detection of the viral nucleic acid.
  • the sample may be collected and may be added to transport medium to stabilize the virus.
  • Nasopharyngeal, nasal and throat swabs may be added to a transport medium.
  • Nasopharyngeal aspirates and washes may or may not be stabilized by addition of transport medium.
  • the virus may be inactivated and lysed to liberate the viral RNA.
  • the nucleic acid may optionally then be extracted to remove potential inhibitors or other interfering agents of later assay steps.
  • viral nucleic acids may be mixed with components essential for specific detection of influenza A, in particular the detection of influenza A of subtype pandemic H1 N1 (2009).
  • the method of detecting the presence of influenza A virus in a biological sample and/or the method of amplifying the nucleic acid of influenza A virus of subtype pandemic H1 N1 may further comprise a step of analysing the amplified sequence.
  • the step of analysis may include sequencing. More in particular, the sequencing step may be a method of pyrosequencing using a sequencing primer. Use of Pyrosequencing directly on clinical material enabled the measurement of the dynamics of these mixed genotypes without the polluting or diluting effect of viral culture.
  • the present invention provides at least one kit for the detection of influenza A of subtype pandemic H1 N1 (2009), the kit comprising at least one oligonucleotide, pair of oligonucleotides or set of oligonucleotides according to any aspect of the present invention.
  • the kit may be used by clinicians to detect human and avian influenza viruses in patients that are afflicted with influenza symptoms.
  • Such kit will include one or more primer and probe sets for the detection of influenza A, of subtype pandemic H1 N1 (2009).

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Abstract

L'invention concerne une méthode de détection de la présence du virus pandémique de la grippe A H1 N1 (2009) dans un échantillon. Cette méthode consiste à détecter la présence d'un amplicon, d'un fragment et/ou d'un dérivé de celui-ci (a) amplifié par une paire d'amorces. L'invention concerne également une/des amorce(s) et/ou sonde, ainsi qu'un kit de détection de la présence de la grippe A H1 N1 (2009) du sous-type pandémique dans un échantillon et/ou chez un patient.
PCT/SG2011/000164 2010-04-26 2011-04-26 Méthodes et/ou amorces de détection de la grippe a WO2011136743A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014066749A3 (fr) * 2012-10-25 2014-08-28 Intelligent Medical Devices, Inc. Sondes et amorces optimisées et procédés d'utilisation de celles-ci pour la liaison, la détection, la différenciation, l'isolement et le séquençage de la grippe a, de la grippe b et du virus syncytial respiratoire

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2011058580A1 (fr) * 2009-11-12 2011-05-19 Defence Research & Development Organisation Oligonucléotides et procédé de détection du virus de la grippe porcine

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WO2011058580A1 (fr) * 2009-11-12 2011-05-19 Defence Research & Development Organisation Oligonucléotides et procédé de détection du virus de la grippe porcine

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014066749A3 (fr) * 2012-10-25 2014-08-28 Intelligent Medical Devices, Inc. Sondes et amorces optimisées et procédés d'utilisation de celles-ci pour la liaison, la détection, la différenciation, l'isolement et le séquençage de la grippe a, de la grippe b et du virus syncytial respiratoire

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