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WO2007058499A1 - Oligonucleotides pour detecter des acides nucleiques de virus respiratoires - Google Patents

Oligonucleotides pour detecter des acides nucleiques de virus respiratoires Download PDF

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WO2007058499A1
WO2007058499A1 PCT/KR2006/004856 KR2006004856W WO2007058499A1 WO 2007058499 A1 WO2007058499 A1 WO 2007058499A1 KR 2006004856 W KR2006004856 W KR 2006004856W WO 2007058499 A1 WO2007058499 A1 WO 2007058499A1
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seq
oligonucleotide
nos
specificity
nucleotide sequences
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Jong Yoon Chun
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Seegene Inc
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Seegene Inc
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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

Definitions

  • the present invention relates to oligonucleotides hybridizable with nucleic acids of respiratory viruses, kits comprising them, and processes for amplifying and detecting viral nucleic acids using them.
  • Human respiratory diseases have been known to be caused by the following viruses: influenza viruses A, B and C (INF A, B and C); human parainfluenza viruses 1, 2, 3 and 4 (hPFV 1, 2, 3 and 4); human respiratory syncytial virus (hRSV); human metapneumoviru (hMPV); human coronavirus OC43 (hCoVOC43) and human coronavirus 229E (hCoV229E); human rhinovirus (hRV); adenovirus (AdV); and human enteroviruses (hEV) (S. Bellau-Pujol, et at., Journal of Virological Methods 126 (2005) 53-63).
  • influenza viruses A, B and C INF A, B and C
  • human parainfluenza viruses 1, 2, 3 and 4 hPFV 1, 2, 3 and 4
  • human respiratory syncytial virus hRSV
  • hMPV human metapneumoviru
  • hCoVOC43 human coronavirus OC43
  • These methods detect target viruses by use of probes hybridizable with viral nucleic acids or primers to specifically amplify virus-originated nucleic acids.
  • probes and primers should be designed to target regions with lower variation frequency.
  • Kits for detecting respiratory viruses by PCR are commercially available and expected to show better detection than conventional cell culture methods and immunoassay.
  • primers are likely not to anneal to viral nucleic acids despite of the existence of viral nucleic acids because of inherent genetic diversity of viruses, thereby producing false negative results.
  • false- positive results are often produced due to non-specific PCR amplification. Accordingly, there remain long-felt needs to develop a novel process for overcoming conventional detection processes and giving more accurate and reliable results.
  • the present inventor has made intensive researches to propose a novel approach to detect respiratory viruses with much higher accuracy in a convenient and rapid manner, and as a result discovered that a multitude of respiratory viruses are accurately detected by use of hybridization oligonucleotides having a unique structure of dual specificity oligonucleotides developed by the present inventor.
  • an object of this invention to provide an oligonucleotide hybridizable specifically with a nuclei acid of a respiratory virus.
  • the present invention relates to oligonucleotides to hybridize specifically with nucleic acid molecules of respiratory viruses.
  • the present invention provides an oligonucleotide hybridizable specifically with a nuclei acid of a respiratory virus, which is represented by the following general formula:
  • X p represents a 5 '-high T n
  • specificity portion having a hybridizing nucleotide sequence substantially complementary to a target sequence to hybridize therewith
  • Y q represents a separation portion comprising at least two universal bases, Z 1 .
  • the separation portion has the lowest T m in the three portions; the separation portion forms a non base-pairing bubble structure under conditions that the 5 '-high T n , specificity portion and the 3 '-low T n , specificity portion are annealed to the target sequence, enabling the 5'-high T n , specificity portion to separate from the 3 '-low T n , specificity portion in terms of hybridization specificity to the target sequence, whereby the hybridization specificity of the oligonucleot
  • the present invention is directed to oligonucleotides hybridizable with a nucleic acid of influenza viruses A and B (INF); human parainfluenza viruses 1, 2 and 3 (hPIV); human respiratory syncytial viruses A and B (hRSV A and B); human metapneumovirus (hMPV); human coronavirus OC43 (hCoVOC43); human coronavirus 229E (hCoV229E); human rhinovirus (hRV); and adenovirus (Adv).
  • the term used herein "nucleic acid of viruses” refers to not only viral genomes but also any RNA or DNA molecule (e.g., cDNA) derived from viral genomes.
  • viral genome is intended to mean the same meaning as nucleic acid of viruses, unless otherwise indicated.
  • hybridization refers to the formation of a double-stranded nucleic acid by base-paring of complementary single stranded nucleic acids. Hybridization may occur between single stranded nucleic acids with some mismatched sequences as well as between single stranded nucleic acids with perfect complementarity. The complementarity for hybridization depends on hybridization conditions, in particular, temperature. Generally, as the hybridization temperature becomes higher, only perfectly complementary sequences are likely to be hybridized; in contrast, as the hybridization temperature becomes lower, hybridization may occurs between single stranded nucleic acids with some mismatched sequences. As the hybridization temperature becomes lower, the mismatch occurs with higher frequency.
  • oligonucleotides having such structure are named as dual specificity oligonucleotides (DSO).
  • DSO dual specificity oligonucleotides
  • the DSO embodies a novel concept and its hybridization is dually determined by the 5 '-high T n , specificity portion and the 3 '-low T n , specificity portion separated by the separation portion, exhibiting dramatically enhanced specificity.
  • the universal base in the separation portion is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2'-deoxyinosine, 2-aza- 2 '-deoxyinosine, 2'-0Me inosine, 2'-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2'-0Me 3- nitropyrrole, 2'-F 3-nitropyrrole, l-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5- nitroindole, 5-nitroindole, 2'-0Me 5-nitroindole, 2'-F 5-nitroindole, deoxy 4- nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4- aminobenzimidazole, deoxy nebularine, 2'-F nebularine, 2'-F 4-nitrobenzimid
  • the universal base or non-discriminatory base analog is deoxyinosine, l-(2'-deoxy-beta-D- ribofuranosyl)-3-nitropyrrole or 5-nitroindole, most preferably, deoxyinosine.
  • the separation portion comprises contiguous nucleotides having universal bases, preferably, deoxyinosine.
  • the 5 '-high T n , specificity portion is longer than the 3 '-low T 1n specificity portion.
  • the 5 '-high T 1n specificity portion is preferably 15-40 nucleotides in length. It is preferable that the 3 '-low T m specificity portion is 3-15 nucleotides in length.
  • the separation portion is preferably 3-10 nucleotides in length.
  • the T m of the 5 '-high T n , specificity portion ranges from 40 0 C to 80 0 C.
  • the T n , of the 3 '-low T m specificity portion ranges preferably from 10 0 C to 40 0 C. It is preferable that the T m of the separation portion ranges from 3°C to 15°C.
  • the respiratory virus-specific oligonucleotide of this invention is constructed (1) to comprise a sequence corresponding or complementary to conserved sequences commonly found among viral isolates; and (2) to have the structure of the dual specificity oligonucleotide.
  • the main reference sequences for preparing oligonucleotides of this invention are conserved sequences selected by searching publicly-known nucleotide sequences of viral isolates. Among the conserved sequences, a sequence suitable to design primers or probes having the DSO structure is then selected.
  • the conserved sequences in respiratory viral genomes may be selected by use of comparing known sequences. For example, the sequence alignment is conducted to find conserved sequences by use of nucleotide sequences as follows: for influenza virus A, GenBank accession Nos. NC_004524 HlNl, NC_004907 H9N2, NC_007363 H5N1, NC_007367 H3N2 and NC_007377 H2N2; for influenza virus B, GenBank accession Nos.
  • the present oligonucleotides embodying the DSO structure with the conserved sequences completely eliminate false-positive results and backgrounds associated with existing methods using conventional primers for detecting respiratory viruses.
  • the important consideration in detection of viruses using primers or probes is genetic diversity.
  • the nucleic acid sequences in even virus isolates of the same species are distinctly different from each other.
  • oligonucleotides are constructed to comprise conserved sequences commonly found in all viral isolates and are hybridized with target viral sequence under low stringent conditions in which hybridization occurs between some mismatched nucleotide sequences.
  • the conventional methods may succeed in reducing false-negative results due to genetic diversity of viruses.
  • low stringent conditions are responsible for the production of non-target products, i.e., false-positive products' and backgrounds because primers or probes are very likely to hybridize with non-target sequences.
  • the present oligonucleotides having the DSO structure is dually determined in terms of their specificity to target sequences with help of the separation portion, they show higher specificity to target sequences even under relatively low stringent conditions.
  • the oligonucleotides of this invention embodying the DSO structure eliminate not only problems of false-negative results due to genetic diversity of respiratory viral sequences but also problems of false-positive results and backgrounds due to hybridization with non-target sequences.
  • the 5'-high T 1n specificity portion and the 3'-low T m specificity separation portion hybridize with a conserved sequence in the target sequence of the viral genome.
  • the separation portion hybridizes with a site showing genetic diversity in the target sequence of the viral genome.
  • the present oligonucleotides having the DSO structure have another advantage in overcoming drawbacks associated with genetic diversity of viral nucleic acids. As demonstrated in Examples, the drawbacks due to genetic diversity of viral nucleic acids are successfully overcome especially when the universal bases of the separation portion in the present oligonucleotides are positioned to correspond to or hybridize with a nucleotide or nucleotides showing genetic diversity.
  • the oligonucleotides of the present invention have additional universal bases in other portions, i.e., the 5 '-high T m specificity portion and/or the 3'- low T n , specificity portion, to correspond to or hybridize with a nucleotide or nucleotides showing genetic diversity.
  • the oligonucleotides of the present invention are designed to comprise a base with genetic diversity. For example, where the nucleic acids of viral isolates have either A or T at a site, two oligonucleotides having either A or T base at the site may be constructed and used.
  • the oligonucleotide of this invention comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 -26.
  • the oligonucleotide of this invention comprises not only the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-26 but also a substantially identical nucleotide sequence to that.
  • substantially identical nucleotide sequence refers to a nucleotide sequence having some deletions, additions and/or substitutions in the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-26. Such nucleotide changes are permissible, so long as the oligonucleotide can be specifically hybridized with a target sequence. It will be appreciated under the doctrine of equivalency that these substantially identical nucleotide sequences fall within the scope of claims.
  • target sequence refers to a nucleotide sequence desired to be hybridized with primers or probes under certain hybridization conditions.
  • target sequence is a sequence indicated in available databases described hereinabove (e.g., GenBank).
  • non-target sequence refers to a nucleotide sequence other than the target sequence.
  • hybridization is performed under high stringent conditions that primers or probes are hybridized with perfectly complementary sequences, only the perfectly complementary sequences are target sequences but sequences with at least one mismatch are non-target sequences or non-specific sequences.
  • products from hybridization with non-target sequences are non-target products or non-specific products.
  • sequences hybridizable with primers or probes are target sequences but other sequences are non-target sequences.
  • the oligonucleotides of this invention serve as probes for detecting target viral nucleic acids.
  • probe means a single- stranded nucleic acid molecule comprising a portion or portions that are substantially complementary to a target nucleotide sequence.
  • Suitable labels include fluorophores, chromophores, chemiluminescers, magnetic particles, radioisotopes, mass labels, electron dense particles, enzymes, cofactors, substrates for enzymes and haptens having specific binding partners, e.g., an antibody, streptavidin, biotin, digoxigenin and chelating group, but not limited to.
  • the labels generate signal detectable by fluorescence, radioactivity, measurement of color development, mass measurement, X-ray diffraction or absorption, magnetic force, enzymatic activity, mass analysis, binding affinity, high frequency hybridization or nanocrystal.
  • the labels may be linked to the 5 '-end, 3 '-end or inner portions of the oligonucleotides.
  • the oligonucleotides may be immobilized on a solid substrate (nitrocellulose membrane, nylon filter, glass plate, silicon wafer and fluorocarbon support) to fabricate microarray.
  • a solid substrate nitrocellulose membrane, nylon filter, glass plate, silicon wafer and fluorocarbon support
  • the present oligonucleotides serve as hybridizable array elements.
  • the immobilization on solid substrates may occur through chemical binding or covalent binding by ultra-violet radiation.
  • the oligonucleotides are bound to a glass surface modified to contain epoxy compounds or aldehyde groups or to a polylysin-coated surface. Furthermore, the oligonucleotides are bound to a substrate through linkers (e.g. ethylene glycol oligomer and diamine).
  • linkers e.g. ethylene glycol oligomer and diamine.
  • the oliogonucleotides of this invention serve as primers for detecting target viral nucleic acids by amplification.
  • the term "primer” as used herein refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand (template) is induced, i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification.
  • the primer is an oligodeoxyribonucleotide.
  • the primer of this invention can be comprised of naturally occurring dNMP (i.e., dAMP, dGM, dCMP and dTMP), modified nucleotide, or non-natural nucleotide.
  • the primer can also include ribonucleotides.
  • the oligonucleotides of this invention may include nucleotides with backbone modifications such as peptide nucleic acid (PNA) (M.
  • PNA peptide nucleic acid
  • nucleotides with sugar modifications such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O- allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides having base modifications such as C-5 substituted pyrimidines (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-,
  • sequences of the primers may comprise some mismatches, so long as they can be hybridized with templates and serve as primers.
  • the nucleic acid amplification of target viral nucleic acids using the oligonucleotides of this invention is used to detect target viruses.
  • an oligonucleotide pair hybridizable specifically with a nuclei acid of a respiratory virus.
  • the preferable oligonucleotide pair comprises each of the nucleotide sequences of SEQ ID NOs: 1 and 2; each of the nucleotide sequences of SEQ ID NOs:3 and 4; each of the nucleotide sequences of SEQ ID NOs:5 and 6; each of the nucleotide sequences of SEQ ID NOs:7 and 8; each of the nucleotide sequences of SEQ ID NOs:9 and 10; each of the nucleotide sequences of SEQ ID NOs: 11 and 12; each of the nucleotide sequences of SEQ ID NOs: 13 and 14; each of the nucleotide sequences of SEQ ID NOs: 15 and 16; each of the nucleotide sequences of SEQ ID NOs: 17 and 18; each of the nucleotide sequences of SEQ ID NOs: 19 and 20; each of the nucleotide sequences of SEQ ID NOs:21 and 22 (each of the nucleotide sequence
  • oligonucleotide pair comprises the oligonucleotides of this invention described hereinabove, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.
  • the oligonucleotide pair is used as a primer pair (forward and reverse primers) for detecting respiratory viruses.
  • an oligonucleotide set hybridizable with nucleic acid molecules of respiratory viruses comprising a least two oligonucleotide pairs described above.
  • the oligonucleotide set comprises the oligonucleotides of this invention described hereinabove, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.
  • the oligonucleotide set is useful not only as a probe set in detecting respiratory viruses but also as a primer set in amplifying nucleic acids of respiratory viruses. If necessary, several types of the present oligonucleotides may be simultaneously used. Such simultaneous application does not influence adversely on either each result or overall results.
  • the oligonucleotide set is used as primer sets for multiplex PCR.
  • the oligonucleotide set is composed of SEQ ID NOs: 5 and 6; SEQ ID NOs: 7 and 8; SEQ ID NOs: 9 and 10; SEQ ID NOs: 15 and 16; SEQ ID NOs: 19 and 20; and SEQ ID NOs: 13 and 14.
  • the oligonucleotide set is composed of SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 11 and 12; SEQ ID NOs: 13 and 14; SEQ ID NOs: 17 and 18; and SEQ ID NOs: 21 and 22 (or SEQ ID NOs: 15 and 26).
  • kits for detecting a respiratory virus or a kit for amplifying a target sequence in a genome sequence of a respiratory virus comprising the oligonucleotides, oligonucleotide pairs or oligonucleotide sets of this invention. Since the kit comprises the oligonucleotides of this invention described hereinabove, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.
  • the present kits may optionally include the reagents required for performing PCR reactions such as buffers, DNA polymerase, DNA polymerase cofactors, and deoxyribonucleotide-5-triphosphates.
  • the kits may also include various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • kits may also include reagents necessary for performing positive and negative control reactions. Optimal amounts of reagents to be used in a given reaction can be readily determined by the skilled artisan having the benefit of the current disclosure.
  • the kits typically, are adapted to contain in separate packaging or compartments the constituents afore-described.
  • a method for detecting a respiratory virus which comprises hybridizing the oligonucleotide, oligonucleotide pair or oligonucleotide set of this invention with a nucleic acid molecule in a respiratory viral genome.
  • a method for amplifying a nucleic acid molecule of a respiratory virus which comprises hybridizing the oligonucleotide, oligonucleotide pair or oligonucleotide set of this invention with a nucleic acid molecule in a respiratory viral genome. More specifically, the present method comprises the steps of: (a) annealing the oligonucleotide as a primer to a nucleic acid molecule in a respiratory viral genome as a template; (b) extending the primer; (c) denaturing the extended product of the step (b) ; and (d) repeating the steps (a)-(c) at least twice.
  • Exemplified primer pair useful in the amplification of this invention includes SEQ ID NO: 1
  • Suitable hybridization conditions may be routinely determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength may be varied depending on various factors, including the length and GC content of oligonucleotide and target nucleotide sequence.
  • the detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.( 1999).
  • the hybridization is performed at temperature of 40-70 0 C, more preferably, 45-68°C, most preferably 50-65 0 C.
  • the present method for amplifying nucleic acids of respiratory viruses may be carried out according to a variety of conventional primer-involving amplification processes.
  • the present method for amplifying nucleic acids may be applied to the amplification of nucleic acids of any respiratory viruses.
  • the nucleic acid molecule may be either DNA or RNA.
  • the molecule may be in either a double-stranded or single-stranded form.
  • the nucleic acid as starting material is double-stranded, it is preferred to render the two strands into a single-stranded or partially single-stranded form.
  • Methods known to separate strands includes, but not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods
  • strand separation can be achieved by heating at temperature ranging from 80 0 C to 105 0 C.
  • General methods for accomplishing this treatment are provided by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001 ).
  • oligonucleotide dT primer hybridizable to poly A tail of mRNA is used.
  • the oligonucleotide dT primer is comprised of dTMPs, one or more of which may be replaced with other dNMPs so long as the dT primer can serve as primer.
  • Reverse transcription can be done with reverse transcriptase that has RNase H activity. If one uses an enzyme having RNase H activity, it may be possible to omit a separate RNase H digestion step by carefully choosing the reaction conditions.
  • the primer used for the present invention is hybridized or annealed to a site on the template such that double-stranded structure is formed.
  • Conditions of nucleic acid annealing suitable for forming such double stranded structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).
  • a variety of DNA polymerases can be used in the amplification step of the present methods, which includes "Klenow" fragment of E. coli DNA polymerase I, a thermostable DNA polymerase, and bacteriophage T7 DNA polymerase.
  • the polymerase is a thermostable DNA polymerase which may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilics (Tth), Thermits filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcw furiosus (Pfu). Many of these polymerases may be isolated from bacterium itself or obtained commercially. Polymerase to be used with the subject invention can also be obtained from cells which express high levels of the cloned genes encoding the polymerase.
  • components required for such reaction When a polymerization reaction is being conducted, it is preferable to provide the components required for such reaction in excess in the reaction vessel. Excess in reference to components of the extension reaction refers to an amount of each component such that the ability to achieve the desired extension is not substantially limited by the concentration of that component. It is desirable to provide to the reaction mixture an amount of required cofactors such as Mg 2+ , dATP, dCTP, dGTP, and dTTP in sufficient quantity to support the degree of the extension desired.
  • the amplification process of the present invention can be done in a single reaction volume without any change of conditions such as addition of reactants.
  • Annealing or hybridization in the present method is performed under stringent conditions that allow for specific binding between the primer and the template nucleic acid (at this time, the separation portion cannot be annealed to the template nucleic acid).
  • stringent conditions for annealing will be sequence-dependent and varied depending on environmental parameters.
  • the annealing temperature ranges from 40 0 C to 70 0 C, more preferably from 45°C to 68°C, most preferably from 50 0 C to 65 0 C.
  • the amplification is performed in accordance with PCR which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.
  • the isolation (or purification) of amplified product may follow the second-stage amplification. This can be accomplished by gel electrophoresis, column chromatography, affinity chromatography or hybridization.
  • the amplified product of this invention may be inserted into suitable vehicle for cloning.
  • the amplified product of this invention may be expressed in suitable host harboring expression vector. In order to express the amplified product, one would prepare an expression vector that carries the amplified product under the control of, or operatively linked to a promoter.
  • the promoter used for prokaryotic host includes, but not limited to, pL ⁇ promoter, trp promoter, lac promoter and T7 promoter.
  • the promoter used for eukaryotic host includes, but not limited to, metallothionein promoter, adenovirus late promoter, vaccinia virus 7.5K promoter and the promoters derived from polyoma, adenovirus 2, simian virus 40 and cytomegalo virus.
  • Certain examples of prokaryotic hosts are E.
  • cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequences.
  • recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequences.
  • recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, tobacco mosaic virus) or transformed with recombinant plasmi
  • the present method is carried out according to multiplex PCR.
  • the results obtained with multiplex PCR are frequently complicated by the artifacts of the amplification procedure. These include “false-negative” results due to reaction failure and “false-positive” results such as the amplification of spurious products, which may be caused by annealing of the primers to sequences which are related to but distinct from the true recognition sequences. Therefore, elaborate optimization steps of multiplex PCR are conducted to reduce such false results; however, the optimization of the reaction conditions for multiplex PCR may become labor-intensive and time-consuming and unsuccessful.
  • the present method amplifies simultaneous a variety of nucleic acid molecules of respiratory viruses with no false results in a single PCR reaction to completely overcome shortcomings associated with conventional multiplex PCR.
  • the present method for amplifying nucleic acid molecules of respiratory viruses comprises (a) annealing at least two oligonucleotides as a primer hybridizable with at least two viral nucleic acids to a nucleic acid molecule in a respiratory viral genome as a template; (b) extending the primer; (c) denaturing the extended product of the step (b) ; and (d) repeating the steps (a)-(c) at least twice.
  • RNA viruses although the genomes of respiratory viruses (mostly, RNA viruses) show genetic diversity, the present oligonucleotides specifically hybridize with a target sequence under relatively low stringent conditions such that they completely overcome problems of false- negative and false-positive products due to genetic diversity; and (b) the present oligonucleotides exhibit dramatic workability in multiplex PCR, enabling to simultaneously detect various respiratory viruses in a single PCR reaction.
  • Figs. Ia-Ib show the results of positive control PCR amplifications.
  • M 100 bp ladder
  • 1 human adenovirus
  • 2 human metapneumovirus
  • 3 human coronavirus 229E
  • 4 human parainfluenza virus 1
  • 5 human parainfluenza virus 2
  • 6 human parainfluenza virus 3
  • Sl mixed sample of 1-6
  • 7 influenza virus A
  • 8 influenza virus B
  • 9 human respiratory syncytial virus B
  • 10 human rhinovirus
  • 11 human respiratory syncytial virus A
  • 12 human coronavirus
  • Fig. Ia primer set I
  • Fig. Ib primer set II
  • Figs. 2a-2c represent the results of multiplex PCR amplifications using primer sets of this invention and viral samples from 24 patients (Fig. 2a: primer set I; Fig. 2b: primer set II; and Fig. 2c: control primer set).
  • N normal sample (negative control); 1-24 (the number of patients); P: positive control;
  • AdV human adenovirus; MPV: human metapneumovirus;
  • CoV229E human coronavirus 229E; PIVl: human parainfluenza virus 1; PIV2: human parainfluenza virus 2; PIV3: human parainfluenza virus 3; INF A: influenza virus A; INF B: influenza virus B; RSV B: human respiratory syncytial virus B; RSV A: human respiratory syncytial virus A; RV: human rhinovirus; CovOC43: human coronavirus OC43.
  • EXAMPLE 1-1 Primers for Amplifying Nucleic Acids of Influenza Virus A (INF A)
  • primers of this invention For designing primers of this invention to successfully amplify all of various genome sequences of influenza virus A showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of influenza virus A. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO (dual specificity oligonucleotide) primers of this invention. In designing the primer sequences, the separation portion (IIIII) of DSO was positioned to a portion corresponding to a region showing genetic diversity.
  • DSO dual specificity oligonucleotide
  • the primers for amplifying influenza virus A were designed to amplify avian influenza viruses (H5N1 and H9N2) as well as human influenza viruses.
  • the numbers indicated below are accession numbers of GenBank in NCBI and the underlines denote sequences showing genetic diversity.
  • NC_OO4524 H1N1 AGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTG--
  • NC 004524 HlNl ATAGCCTTAGCTGTAGTGCTGGCTAAAACCATTCTGTT—
  • NC_004907 H9N2 ATAGCCTTAGCTGTAGTGCTGGCTAGTACCATTCTGTT— NC 007363 H5N1 — ATAGCCTTAGCTGTAGTGCTGGCCAGCACCATTCTGTT—
  • NC 007367 H3N2 ATAGCCTTAGCTGTAGTGCTGGCTAAAACCATTCTGTT—
  • NC 007377 H2N2 AT AGCCTT AGCTGTAGTGCTGGCTAAAACCATTCTGTT— INFA-R598 primer 5'-ATAGCCTTAGCTGTAGTGCTGGCIIIIICCATTCTGTT-S ' (SEQ ID NO: 2)
  • EXAMPLE 1-2 Primers for Amplifying Nucleic Acids of Influenza Virus B (INF B) For designing primers of this invention to successfully amplify all of various genome sequences of influenza virus B showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of influenza virus B. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention. In designing the primer sequences, the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity. Besides the separation portion, "I" was incorporated into positions corresponding to regions showing genetic diversity.
  • EXAMPLE 1-3 Primers for Amplifying Nucleic Acids of Human Parainfluenza Virus 1 (hPIV 1) For designing primers of this invention to successfully amplify all of various genome sequences of hPIV 1 showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hPIV 1. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention. In designing the primer sequences, the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity.
  • EXAMPLE 1-4 Primers for Amplifying Nucleic Acids of Human Parainfluenza Virus 2 (hPIV 2) For designing primers of this invention to successfully amplify all of various genome sequences of hPIV 2 showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hPIV 2. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention. In designing the primer sequences, the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity.
  • EXAMPLE 1-5 Primers for Amplifying Nucleic Acids of Human Parainfluenza Virus 3 (hPIV 3)
  • primers of this invention For designing primers of this invention to successfully amplify all of various genome sequences of hPIV 3 showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hPIV 3. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention. In designing the primer sequences, the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity. Besides the separation portion, "I" was incorporated into positions corresponding to regions showing genetic diversity.
  • hPIV-3-5 1 S'-TAAGIATAAAATGGACATGGCATAAIIIIITATCAAGACC-S' (SEQ ID NO:9)
  • hPIV-3-3 1 5'-CGTTTACICTTTCIGTTGCTGTTGAGIIIIITATGACTGGG-S '(SEQ ID NO: 10)
  • EXAMPLE 1-6 Primers for Amplifying Nucleic Acids of Human Respiratory Syncytial Virus A (hRSVA)
  • primers of this invention For designing primers of this invention to successfully amplify all of various genome sequences of hRSV A showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hRSV A. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention. In designing the primer sequences, the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity.
  • RSVa-F 103 5'-AGAATTTTATCAATCAACATGCAGTGIIIIIAGCAAAGGCT-S ' (SEQ ID NO:11)
  • RSVa-R382 5'-ATTGTTGAGTGTATAATTCATAAACCTTGGIIIIICTCTTCTGGC-
  • EXAMPLE 1-7 Primers for Amplifying Nucleic Acids of Human Respiratory Syncytial Virus B (hRSV B)
  • hRSV B Human Respiratory Syncytial Virus B
  • forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention.
  • the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity.
  • RSVb-F536 5'-TCAGTCTATCAAATGGGGTCAGTGIIIIIACCAGCAAAG-S ' (SEQ ID NO:13)
  • EXAMPLE 1-8 Primers for Amplifying Nucleic Acids of Human Metapneumovirus (hMPV)
  • primers of this invention For designing primers of this invention to successfully amplify all of various genome sequences of hMPV showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hMPV. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention.
  • the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity. Besides the separation portion, "I” was incorporated into positions corresponding to regions showing genetic diversity.
  • the symbol "W” denotes A or T.
  • EXAMPLE 1-9 Primers for Amplifying Nucleic Acids of Human Coronavirus OC43 (hCoVOC43)
  • primers of this invention For designing primers of this invention to successfully amplify all of various genome sequences of hCoVOC43 showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hCoVOC43. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention. In designing the primer sequences, the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity.
  • hCoVOC43-F400 5'-TATGTTAGGCCGATAATTGAGGACTIIIIIACTCTGACGG-3'(SEQ ID NO: 17)
  • primers of this invention For designing primers of this invention to successfully amplify all of various genome sequences of hCoV229E showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hCoV229E. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare
  • DSO was positioned to a portion corresponding to a region showing genetic diversity. Besides the separation portion, "I" was incorporated into positions corresponding to regions showing genetic diversity.
  • EXAMPLE 1-11 Primers for Amplifying Nucleic Acids of Human Rhinovirus (hRV) For designing primers of this invention to successfully amplify all of various genome sequences of hRV showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hRV. Forward and reverse primers were designed with referring to sequences among the conserved sequences suitable to prepare DSO primers of this invention. In designing the primer sequences, the separation portion of DSO was positioned to a portion corresponding to a region showing genetic diversity. The results indicated in Figs. 1-2 were obtained using the primers of SEQ ID NOs:36 and 26.
  • RSVa-F 103C 5 '-AGAATTTTATCAATCAACATGCAGTGCAGTCAGCAAAGGCT-S '
  • RSVa-R382C 5'-ATTGTTGAGTGTATAATTCATAAACCTTGGTAGTTCTCTTCTGG -3'
  • RSVb-F536C 5 '-TCAGTCTATCAAATGGGGTCAGTGTTTTAACCAGCAAAG-S '
  • RSVb-R927C 5'-ATTACACCATAGATAGGTAGCTGTACAACATATGC AAGGACTTC-
  • hCoVOC43-F400C 5 '-TATGTTAGGCCGATAATTGAGGACTACTTTACTCTGACGG-S ' hCoVOC43-3'C: 5'-GTAATTACCGACTTTGGACTTAACATAAACAGCAAAACCAC-S'
  • RNAzol B method From nasal aspirate of patients infected with respiratory viruses, total RNA samples of viruses were extracted using RNAzol B method (RNAzol LS; Tel-Test, Inc.).
  • Reverse transcription reaction was performed using the total RNAs obtained in Example II for 1.5 hr at 37 ° C in a reaction volume of 20 ⁇ l.
  • the reaction contains 5 ⁇ l of total RNA (about 0.5 ⁇ g), 4 ⁇ l of 5 x reaction buffer (Invitrogen, USA), 5 ⁇ l of dNTPs (each 2 mM), 2 ⁇ l of 1 ⁇ M random hexadioxynucleotide, 0.5 ⁇ l of RNase inhibitor (40 units/ ⁇ l, Promega) and 1 ⁇ l of Moloney murine leukemia virus reverse transcriptase (M-MLV, 200 units/ ⁇ l, Promega).
  • M-MLV Moloney murine leukemia virus reverse transcriptase
  • EXAMPLE III-2 Multiplex PCR Multiplex PCR was conducted using cDNA products obtained Example III-l and primer sets described below: (1) Primer set I: primer pairs for amplifying genomes of AdV, MPV, CoV229E, PIV 1, PIV 2 and PIV 3;
  • Primer set II primer pairs for amplifying genomes of INF A, INF B, RSV A, RSV B, RV and CoVOC43.
  • the multiplex PCR amplification was conducted in a single tube by use of the primer set I or II; the reaction mixture was in the final volume of 20 ⁇ l containing 2 ⁇ l (30 ng) of the first strand cDNA, 2 ⁇ l of 10 x PCR reaction buffer containing 15 mM MgCl 2 (Roche), 2 ⁇ l of dNTP (2 mM each dATP, dCTP, dGTP and dTTP), 4 ⁇ l of the primer set I or II and 0.5 ⁇ l of Taq polymerase (5 units/ ⁇ l).
  • the tube containing the reaction mixture was placed in a preheated (94 0 C) thermal cycler and the amplifications were then performed under the following thermal conditions: denaturation at 94°C for 15 min followed by 30-45 cycles of 94°C for 30 sec, 60- 65°C for 1.5 min and 72°C for 1.5 min; followed by a 10-min final extension at 72°C.
  • PCR products were resolved by electrophoresis on an agarose gel containing EtBr and the bands formed were eluted, followed by sequencing.
  • the multiplex PCR amplifications were carried out according to the same processes and conditions as described above, except that the conventional primers (control primers) were used instead of the primer set II.
  • EXAMPLE IV Positive Control PCR Viral RNA samples were obtained from cultures of influenza viruses A and B; human parainfluenza viruses 1, 2 and 3; human respiratory syncytial viruses A and B; human metapneumoviruses; human coronaviruses OC43 and 229E; and adenovirus. Using each viral RNA sample, reverse transcription was conducted according to similar processes to Example III- 1. Then, PCR amplification was performed using each or mixture of the reverse transcription products as templates and the primer sets I and II of Example II-2. PCR products were resolved by electrophoresis on an agarose gel containing EtBr.
  • Fig. 1 shows the results of positive control PCR amplifications, demonstrating that the primers of this invention produce amplicons with expected size.
  • Fig. 2 represents the results of multiplex PCR using primer sets of this invention and viral samples from 24 patients. As shown in Figs. 2a (primer set I) and 2b (primer set II), the primer sets of this invention perfectly detect and identify the type of viruses infecting the patients infected with respiratory viruses.
  • Fig. 2c represents the results of multiplex PCR using the conventional primer set prepared in Example 1-13 instead of the primer set II. As indicated in Fig. 2c, the conventional primers generate several non-target products and backgrounds.
  • the oligonucleotides of this invention are very useful as probes in microarray-based technologies as well as primers in PCR-based technologies.
  • the present invention provides oligonucleotides hybridizable with nucleic acid targets of respiratory viruses, kits comprising them, and processes for amplifying and detecting viral nucleic acids using them.
  • the oligonucleotides of this invention are designed to have the unique structure of the dual specific oligonucleotide (DSO) with referring to conserved sequences of nucleic acids of viral isolates.
  • DSO dual specific oligonucleotide
  • the oligonucleotides of this invention hybridizes specifically with target nucleic acids of respiratory viruses and therefore are very useful in detecting respiratory viruses.

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Abstract

L'invention concerne des oligonucléotides pouvant être hybridés avec des acides nucléiques de virus respiratoires, des kits les contenant et des procédés pour amplifier et détecter des acides nucléiques viraux au moyen de ces oligonucléotides. Les oligonucléotides de l'invention suppriment les problèmes liés aux produits faux négatifs et faux positifs dans la détection de virus respiratoires au moyen d'amorces classiques et ils présentent une aptitude extraordinaire à la transformation par PCR multiplexe, permettant de détecter simultanément plusieurs virus respiratoires lors d'une seule réaction PCR.
PCT/KR2006/004856 2005-11-18 2006-11-17 Oligonucleotides pour detecter des acides nucleiques de virus respiratoires Ceased WO2007058499A1 (fr)

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EP2178897A4 (fr) * 2007-07-17 2011-01-26 Univ Laval Séquences d'acide nucléique pour l'amplification et la détection de virus respiratoires
WO2011027966A3 (fr) * 2009-09-03 2011-05-19 Seegene, Inc. Sonde td et ses utilisations
CN102782150A (zh) * 2009-11-07 2012-11-14 Seegene株式会社 利用靶杂交及检测引物进行靶检测
JP2012532627A (ja) * 2009-07-13 2012-12-20 エイジェンシー フォー サイエンス，テクノロジー アンド リサーチ インフルエンザ検出方法およびそのためのキット
WO2013041853A1 (fr) 2011-09-19 2013-03-28 Epistem Limited Sonde à spécificité de multiples régions cibles et caractère tripartite
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CN110408614A (zh) * 2019-07-22 2019-11-05 深圳市人民医院 用于五种呼吸道病毒检测的试剂盒及其应用
CN113186348A (zh) * 2021-05-21 2021-07-30 广东科蓝生物技术有限公司 检测常见呼吸道病毒的引物组合及方法

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2178897A4 (fr) * 2007-07-17 2011-01-26 Univ Laval Séquences d'acide nucléique pour l'amplification et la détection de virus respiratoires
JP2012532627A (ja) * 2009-07-13 2012-12-20 エイジェンシー フォー サイエンス，テクノロジー アンド リサーチ インフルエンザ検出方法およびそのためのキット
KR101481004B1 (ko) 2009-09-03 2015-01-22 주식회사 씨젠 Ｔｄ프로브 및 그의 용도
WO2011027966A3 (fr) * 2009-09-03 2011-05-19 Seegene, Inc. Sonde td et ses utilisations
WO2011028041A3 (fr) * 2009-09-03 2011-09-15 Seegene, Inc. Sonde td et ses utilisations
US12371736B2 (en) 2009-09-03 2025-07-29 Seegene, Inc. TD probe and its uses
CN102782150B (zh) * 2009-11-07 2015-04-01 Seegene株式会社 利用靶杂交及检测引物进行靶检测
AU2009355009B2 (en) * 2009-11-07 2014-05-08 Seegene, Inc. THD primer target detection
AU2009355009A8 (en) * 2009-11-07 2014-05-22 Seegene, Inc. THD primer target detection
EP2496709A4 (fr) * 2009-11-07 2013-03-27 Seegene Inc Détection ciblée par amorce thd
JP2013509871A (ja) * 2009-11-07 2013-03-21 シージーン アイエヌシー Ｔｈｄプライマーターゲット検出
CN102782150A (zh) * 2009-11-07 2012-11-14 Seegene株式会社 利用靶杂交及检测引物进行靶检测
WO2013041853A1 (fr) 2011-09-19 2013-03-28 Epistem Limited Sonde à spécificité de multiples régions cibles et caractère tripartite
RU2506317C2 (ru) * 2012-04-17 2014-02-10 Российская Федерация, от имени которой выступает Министерство промышленности и торговли Российской Федерации (Минпромторг России) Способ выявления кишечных вирусов в клинических образцах и воде методом мультиплексной пцр с детекцией в режиме реального времени и перечень последовательностей для его осуществления
CN110408614A (zh) * 2019-07-22 2019-11-05 深圳市人民医院 用于五种呼吸道病毒检测的试剂盒及其应用
CN113186348A (zh) * 2021-05-21 2021-07-30 广东科蓝生物技术有限公司 检测常见呼吸道病毒的引物组合及方法

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