WO2007058499A1

WO2007058499A1 - Oligonucleotides for detecting respiratory virus nucleic acids

Info

Publication number: WO2007058499A1
Application number: PCT/KR2006/004856
Authority: WO
Inventors: Jong Yoon Chun
Original assignee: Seegene, Inc.
Priority date: 2005-11-18
Filing date: 2006-11-17
Publication date: 2007-05-24
Also published as: KR20070052890A

Abstract

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. The present oligonucleotides completely overcome problems of false-negative and false-positive products in detection of respiratory viruses using conventionalxprimers and show dramatic workability in multiplex PCR, enabling to simultaneously detect various respiratory viruses in a single PCR reaction.

Description

OLIGONUCLEOTIDES FOR DETECTING RESPIRATORY VIRUS NUCLEIC ACIDS

FIELD OF THE INVENTION

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.

DESCRIPTION OFTHE RELATEDART

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). For detecting viruses, various processes such as method using cell culture and method to detect viral antigens or antibodies have been proposed (Gardner, P.S., et al, Br. Med. J. 3 (614), 340-343). However, such methods fail to satisfy several important considerations such as assay time, convenience and specificity; therefore, there remains a need to develop novel processes for overcoming shortcomings associated with conventional methods. In recent, several approaches using nucleic acid hybridization have been proposed to detect viral genomes or RNA or DNA molecules derived from them.

These methods detect target viruses by use of probes hybridizable with viral nucleic acids or primers to specifically amplify virus-originated nucleic acids.

In the meantime, it has been well known that viruses have a multitude of variants due to their genetic diversity and their nucleic acids are generally contained in a very low level. In this regard, it becomes pivotal that probes or primers are designed to show higher specificity and sensitivity in processes using nucleic acid hybridization.

It has been reported that the variation in viral genomes depends on nucleotide sequence regions. Therefore, 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. However, in the processes conducted according to PCR, 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. To make matters worse, 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.

Throughout this application, various patents and publications are referenced, and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVETNION

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.

Accordingly, it is an object of this invention to provide an oligonucleotide hybridizable specifically with a nuclei acid of a respiratory virus.

It is another object of this invention to provide a kit for detecting or amplifying a nucleic acid molecule of a respiratory virus.

It is still another object of this invention to provide a method for detecting or amplifying a nucleic acid molecule of a respiratory virus.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

The present invention relates to oligonucleotides to hybridize specifically with nucleic acid molecules of respiratory viruses.

More specifically, the present invention provides an oligonucleotide hybridizable specifically with a nuclei acid of a respiratory virus, which is represented by the following general formula:

5'-X_p-Y_q-Z_r-3' wherein, 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, Z₁. represents a 3 '-low T_m specificity portion having a hybridizing nucleotide sequence substantially complementary to a target sequence to hybridize therewith, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; the T_n, of the 5 '-high T_n, specificity portion is higher than that of the 3 '-low T_n, specificity portion, 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 oligonucleotide is determined dually by the 5 '-high T_n, specificity portion and the 3 '-low T_n, specificity portion such that the overall hybridization specificity of the oligonucleotide is enhanced; wherein the respiratory virus is influenza virus, human parainfluenza virus, human respiratory syncytial virus, human metapneumovirus, human coronavirus, human rhinovirus or adenovirus; and wherein the target sequence is a genome sequence of the respiratory virus. 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. Furthermore, the term used herein "viral genome" is intended to mean the same meaning as nucleic acid of viruses, unless otherwise indicated.

The term used "hybridization" herein 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.

The fundamental structure of oligonucleotides of this invention has been first proposed by the present inventor and called as a structure with dual specificity. Therefore, oligonucleotides having such structure are named as dual specificity oligonucleotides (DSO). 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.

According to a preferred embodiment, 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-nitrobenzimidazole, PNA-5- introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4- nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4- nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethyl inosine, 2O- methoxyethyl nebularine, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitrobenzimidazole, 2'-0-methoxyethyl 3-nitropyrrole, and combinations thereof. More preferably, the universal base or non-discriminatory base analog is deoxyinosine, l-(2'-deoxy-beta-D- ribofuranosyl)-3-nitropyrrole or 5-nitroindole, most preferably, deoxyinosine.

It is preferable that the separation portion comprises contiguous nucleotides having universal bases, preferably, deoxyinosine.

Preferably, 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.

According to a preferred embodiment, the T_mof the 5 '-high T_n, specificity portion ranges from 40⁰C to 80⁰C. The T_n, of the 3 '-low T_m specificity portion ranges preferably from 10⁰C to 40⁰C. It is preferable that the T_m of the separation portion ranges from 3°C to 15°C.

According to a preferred embodiment, 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. AF101993, AJ781171, AY044173, AF101999, AFl 02001, AF102003, AY582055, AY582056, AY504600, AY687393, AY582047, AY582051, AJ781174, AY582052 and AY582053; for human respiratory syncytial virus A, GenBank accession Nos. AY526553, HRSVFG, RSHFUSP, PNRSFO, HRU31559, NCJ)Ol 803 and RSH22K; for human respiratory syncytial virus B, GenBank accession Nos. AF013254, AF013255, AY353550 D00334, AY526558, AY526559, AY526563, AY526566, AY526561, AY526560, AY526562 and AY526567; human coronavirus 229E, GenBank accession Nos., hcorona 229e hcorona nl63 (22879); human coronavirus OC43, GenBank accession Nos., gi50844468, gi329574, gi37702815, gi50844477, gi50844468 and gi34398245; for human metapneumovirus, GenBank accession Nos., gi42632384, gi42632366, gi42632357, gi56411384, gi42632384 and gi42632366; human parainfluenza virus 1, GenBank accession Nos., gil621406, gil621414, gil621404, gil621410, gil621408, gi332674, gi332673, gi332672, gi332676 and gi332677; for human parainfluenza virus 2, GenBank accession Nos., AB176531, AF533011, AF533012, PAFPIV2HN and SNDNPPMF; for human parainfluenza virus 3, GenBank accession Nos., AB 189960, AB 189961, PIFHNA, PIFHNE and PIFHNLA; for human rhinovirus, GenBank accession Nos. NC_001617 NCJ)0149; for human adenovirus, HAdV typel, HAdV type2, HAdV type5, HAdV E and HAdV B.

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.

According to conventional methods, 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.

Therefore, the conventional methods may succeed in reducing false-negative results due to genetic diversity of viruses. However, 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.

In contrast, where the hybridization is performed under higher stringent conditions to overcome problems of false-positive products and backgrounds, the false-negative results are likely to produce. However, since 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.

According to a preferred embodiment, 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. According to a preferred embodiment, 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.

According to a preferred embodiment, 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. According to a preferred embodiment, 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.

Most preferably, the oligonucleotide of this invention comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 -26.

The nucleotide sequences of SEQ ID NOs: 1-26 and their target viruses are summarized in Table 1. TABLE 1

^* I: deoxyinosine; R: A or G; W: A or T; and Y: C or T

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. The term used herein "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.

The term used herein "target sequence" refers to a nucleotide sequence desired to be hybridized with primers or probes under certain hybridization conditions. Preferably, the target sequence is a sequence indicated in available databases described hereinabove (e.g., GenBank).

The term used herein "non-target sequence" refers to a nucleotide sequence other than the target sequence. For example, where 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. Furthermore, products from hybridization with non-target sequences are non-target products or non-specific products. Unlikely, where hybridization is performed under low stringent conditions that primers or probes are hybridized with perfectly and imperfectly complementary sequences with some mismatches, sequences hybridizable with primers or probes are target sequences but other sequences are non-target sequences.

In one embodiment of this invention, the oligonucleotides of this invention serve as probes for detecting target viral nucleic acids. The term used herein "probe" means a single- stranded nucleic acid molecule comprising a portion or portions that are substantially complementary to a target nucleotide sequence. Some modifications in the oliogonucleotides of this invention can be made unless the modifications abolish the advantages of the oliogonucleotides. Such modifications, i.e., labels linking to the oliogonucleotides generate a signal to detect hybridization. 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. Labeling may be performed directly (e.g., with dyes) or indirectly (e.g., with biotin, digoxin, alkaline phosphatase or horseradish peroxidase). According to a preferred embodiment, the oligonucleotides may be immobilized on a solid substrate (nitrocellulose membrane, nylon filter, glass plate, silicon wafer and fluorocarbon support) to fabricate microarray. In microarray, 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. In an embodiment of this invention, 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).

According to one embodiment of this invention, 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. Preferably, 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. For instance, the oligonucleotides of this invention For example, the oligonucleotide of this invention may include nucleotides with backbone modifications such as peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365:566-568(1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, 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-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-), 7-deazapurines with C-7 substituents (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine, and diaminopurine.

The sequences of the primers may comprise some mismatches, so long as they can be hybridized with templates and serve as primers.

In one embodiment of this invention, the nucleic acid amplification of target viral nucleic acids using the oligonucleotides of this invention is used to detect target viruses.

In another aspect of this invention, there is provided 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 sequences of SEQ ID NOs:25 and 26); or each of the nucleotide sequences of SEQ ID NOs:23 and 24..

Since the 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.

Preferably, the oligonucleotide pair is used as a primer pair (forward and reverse primers) for detecting respiratory viruses.

In still another aspect of this invention, there is provided an oligonucleotide set hybridizable with nucleic acid molecules of respiratory viruses comprising a least two oligonucleotide pairs described above.

Since 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.

According to a preferred embodiment, the oligonucleotide set is used as primer sets for multiplex PCR.

According to a preferred embodiment, 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.

According to a preferred embodiment, 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).

In further aspect of this invention, there is provided a kit 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. Optionally, the kits may also include various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.

The 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.

In still further aspect of this invention, there is provided 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.

In another aspect of this invention, there is provided 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

NOs: 1 and 2; SEQ ID NOs:3 and 4; SEQ ID NOs:5 and 6; SEQ ID NOs:7 and 8; SEQ ID

NOs:9 and 10; SEQ ID NOs: 11 and 12; SEQ ID NOs: 13 and 14; SEQ ID NOs: 15 and 16; SEQ

ID NOs: 17 and 18; SEQ ID NOs: 19 and 20; SEQ ID NOs:21 and 22; SEQ ID NOs:23 and 24; or SEQ ID NOs:25 and 26.

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).

According to a preferred embodiment, the hybridization is performed at temperature of 40-70⁰C, more preferably, 45-68°C, most preferably 50-65⁰C.

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. Where 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

(e.g., helicase action), and binding proteins. For instance, strand separation can be achieved by heating at temperature ranging from 80⁰C to 105⁰C. 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 ).

Where a mRNA is employed as starting material, a reverse transcription step is necessary prior to performing annealing step, details of which are found in Joseph Sambrook, et al.,

Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, N.Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988)). For reverse transcription, an 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. Preferably, 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.

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 Mg²⁺, dATP, dCTP, dGTP, and dTTP in sufficient quantity to support the degree of the extension desired.

All of the enzymes used in this amplification reaction may be active under the same reaction conditions. Indeed, buffers exist in which all enzymes are near their optimal reaction conditions. Therefore, 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). Such stringent conditions for annealing will be sequence-dependent and varied depending on environmental parameters. Preferably, the annealing temperature ranges from 40⁰C to 70⁰C, more preferably from 45°C to 68°C, most preferably from 50⁰C to 65⁰C.

In the most preferable embodiment, 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 present methods may be combined with many other processes known in the art to achieve a specific aim. For example, 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. In addition, the amplified product of this invention may be inserted into suitable vehicle for cloning. Furthermore, 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. Many standard techniques are available to construct expression vectors containing the amplified product and transcriptional/translational/control sequences in order to achieve protein or peptide expression in a variety of host-expression systems. The promoter used for prokaryotic host includes, but not limited to, pLλ 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. coli, Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. In addition to microorganisms, 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. In addition to mammalian cells, 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. The expressed polypeptide from the amplified product may be generally purified with a variety of purposes in accordance with the method known in the art.

According to a preferred embodiment, the present method is carried out according to multiplex PCR. However, 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.

Where the present method for amplifying nucleic acid molecules of respiratory viruses is carried out according to multiplex PCR, it 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. The advantages of this invention are will be described as follows:

(a) 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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

OC43, S2: mixed sample of 7-11 (Fig. Ia: primer set I; and 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.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples. EXAMPLE EXAMPLE I: Primer Design and Preparation

Conserved sequences were discovered by comparing the nucleotide sequences of virus isolates and suitable sequences to design primers of this invention were selected among them. The symbol "I" denotes deoxyinosine in the following sequences.

EXAMPLE 1-1 : Primers for Amplifying Nucleic Acids of Influenza Virus A (INF A)

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.

In particular, 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_004907 H9N2 — AGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTG— NC_007363 H5N1 — AGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTG— NC_007367 H3N2 — AGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTG— NC_007377 H2N2 — AGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTG— INFA-F85 primer 5 '-AGGCCCCCTCAAAGCCGAGAIIIIICAGAGACTTG-S' (SEQ ID

NO: 1)

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.

INFB-5'-955 5'-TTGGCTATGACIGAAAGIATAACCIIIIICAGCCCAA-S ' (SEQ ID NO:3) INFB-3'-1409 5'-TTACATGTTCGGTAAAAITCGTTTAIIIIITCCATACATG-S ' (SEQ ID

NO:4)

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.

PIVl-HN-Fl 5'-GACCCACATGATTTCTGGAGATGIIIIITAGGAGAACC-3¹ (SEQ ID NO:5)

PIVl-HN-Rl 5'-CTATTACAGAACATGATTTCCTGTTGTCIIIIITGTCATAGGT-S' (SEQ ID NO:6)

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. hPIV-2-5' 5'-TGGACCTCTCCTAAATATICCCAGCIIIIICCCCTCAGCA-S XSEQ ID NO:7) hPIV-2-3' 5'-GTGACTGAACAGCTTTTGCGATTGIIIIIATCACTTAGG-S ' (SEQ ID NO:8)

EXAMPLE 1-5: Primers for Amplifying Nucleic Acids of Human Parainfluenza Virus 3 (hPIV 3)

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-3-5¹ S'-TAAGIATAAAATGGACATGGCATAAIIIIITATCAAGACC-S' (SEQ ID NO:9) hPIV-3-3¹ 5'-CGTTTACICTTTCIGTTGCTGTTGAGIIIIITATGACTGGG-S '(SEQ ID NO: 10)

EXAMPLE 1-6: Primers for Amplifying Nucleic Acids of Human Respiratory Syncytial Virus A (hRSVA)

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-

3'(SEQ ID NO: 12)

EXAMPLE 1-7: Primers for Amplifying Nucleic Acids of Human Respiratory Syncytial Virus B (hRSV B) For designing primers of this invention to successfully amplify all of various genome sequences of hRSV B showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of hRSV 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.

RSVb-F536 5'-TCAGTCTATCAAATGGGGTCAGTGIIIIIACCAGCAAAG-S ' (SEQ ID NO:13)

RS Vb-R927 5 '-ATTAC ACC ATAGAT AGGTAGCTGT AC AACIIIIIC AAGGACTTC-3 ' (SEQ ID NO: 14)

EXAMPLE 1-8: Primers for Amplifying Nucleic Acids of Human Metapneumovirus (hMPV)

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. 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. The symbol "W" denotes A or T. hMPV-5'-585 5'-AGCTTCAGTCAATTCAACAGAAIIIIICTAAATGTTG-S ' (SEQ ID NO:15) hMPV-3'-1007 5'-TTGAITGCTCAGCIACATTAATIIIIICWGCTGTGTC-S ' (SEQ ID NO: 16)

EXAMPLE 1-9: Primers for Amplifying Nucleic Acids of Human Coronavirus OC43 (hCoVOC43)

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) hCoVOC43-3' 5'-GTAATTACCGACTTTGGACTTAACATIIIIIGCAAAACCAC-S XSEQ ID NO: 18)

EXAMPLE 1-10: Primers for Amplifying Nucleic Acids of Human Coronavirus 229E (hCoV229E)

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 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.

229E-F375 S'-TICTTAAGCAGTATACTTCTGCTTGTIIIICTATTGAAGA^ '(SEQ ID NO: 19)

229E-R375 S'-TGGCCATICGTTCAGCATCAGCGAIIIIIGGCAAAACCA-S' (SEQ ID NO:20)

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.

HRV-F 151 5'-CCCCACGGGTGACCGTGTCCCAIIIIICGTGGCGGCC-S ' (SEQ ID NO:21) HRV-R151 5'-GCTCCAGGGTTAAGGTTAGCCGCIIIIIGGGGCCGG-S' (SEQ ID

NO:22)

HRVa-F 15 5'-ATCGTTAICCGCAAAGTGCCTIIIIIAAGCYTAGTA-S '(SEQ ID NO:26) HRV-R353 5'-GAAACACGGACACCCAAAGTIIIIIGTCCCRTCCC-S ' (SEQ ID NO:25)

EXAMPLE 1-12: Primers for Amplifying Nucleic Acids of Adenovirus (Adv)

For designing primers of this invention to successfully amplify all of various genome sequences of Adv showing genetic diversity, conserved sequences were discovered by comparing publicly-known nucleotide sequences of Adv. 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. The symbol "R" denotes A or G. HAdV-Fl 5'-TCTAIGGCATCTCGATCCAGCAIIIIICCTCGTTTCG-S ' (SEQ ID

NO:23)

HAdV-Rl 5'-GGACACIIGCTCATGGAIACCARAGIIIIIAAACGCATCAA-S XSEQ ID NO:24)

EXAMPLE 1-13: Preparation of Conventional Primers

With referring to the selected sequences, conventional primers were constructed to have no the DSO structure and used as control primers. INFA-F85C S'-AGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTG-S' INFA-R85C 5'-ATAGCCTTAGCTGTAGTGCTGGCTAAAACCATTCTGTT-S'

INFB-5'-955C 5'-TTGGCTATGACTGAAAGAATAACCAGAGACAGCCCAA-S ' INFB-3'-1409C 5'-TTACATGTTCGGTAAAAGTCGTTTATTCCTTCCATACATG-S '

RSVa-F 103C: 5 '-AGAATTTTATCAATCAACATGCAGTGCAGTCAGCAAAGGCT-S ' RSVa-R382C: 5'-ATTGTTGAGTGTATAATTCATAAACCTTGGTAGTTCTCTTCTGG -3'

RSVb-F536C: 5 '-TCAGTCTATCAAATGGGGTCAGTGTTTTAACCAGCAAAG-S '

RSVb-R927C: 5'-ATTACACCATAGATAGGTAGCTGTACAACATATGC AAGGACTTC-

3'

hCoVOC43-F400C: 5 '-TATGTTAGGCCGATAATTGAGGACTACTTTACTCTGACGG-S ' hCoVOC43-3'C: 5'-GTAATTACCGACTTTGGACTTAACATAAACAGCAAAACCAC-S'

EXAMPLE II: Preparation of Viral Nucleic Acids

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.).

EXAMPLE in: RT-PCR and Multiplex PCR EXAMPLE III-l: RT-PCR (reverse transcriptase-PCR)

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 μl. The reaction contains 5 μl of total RNA (about 0.5 μg), 4 μl of 5 x reaction buffer (Invitrogen, USA), 5 μl of dNTPs (each 2 mM), 2 μl of 1 μM random hexadioxynucleotide, 0.5 μl of RNase inhibitor (40 units/μl, Promega) and 1 μl of Moloney murine leukemia virus reverse transcriptase (M-MLV, 200 units/μl, Promega).

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;

(2) 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 μl containing 2 μl (30 ng) of the first strand cDNA, 2 μl of 10 x PCR reaction buffer containing 15 mM MgCl₂ (Roche), 2 μl of dNTP (2 mM each dATP, dCTP, dGTP and dTTP), 4 μl of the primer set I or II and 0.5 μl of Taq polymerase (5 units/μl). The tube containing the reaction mixture was placed in a preheated (94⁰C) 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. In addition, 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.

Expected sizes of PCR products using the primer set I or II are summarized in Table 2. TABLE 2

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.

For detecting respiratory viruses, 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. The oligonucleotides of this invention hybridizes specifically with target nucleic acids of respiratory viruses and therefore are very useful in detecting respiratory viruses.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. An oligonucleotide hybridizable specifically with a nuclei acid of a respiratory virus, which is represented by the following general formula:

5'-X_p-Y_q-Z_r-3' wherein, X_p represents a 5 '-high T_m 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, Z₁ represents a 3 '-low T_m specificity portion having a hybridizing nucleotide sequence substantially complementary to a target sequence to hybridize therewith, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; the T_n, of the 5'-high T_m specificity portion is higher than that of the 3 '-low T_m specificity portion, 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_m specificity portion are annealed to the target sequence, enabling the 5 '-high T_m 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 oligonucleotide is determined dually by the 5 '-high T_1n specificity portion and the 3 '-low T_m specificity portion such that the overall hybridization specificity of the oligonucleotide is enhanced; wherein the respiratory virus is influenza virus, human parainfluenza virus, human respiratory syncytial virus, human metapneumovirus, human coronavirus, human rhinovirus or adenovirus; and wherein the target sequence is a genome sequence of the respiratory virus.

2. The oligonucleotide according to claim 1, wherein the universal base is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, T- OMe 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-nitrobenzimidazole, PNA-5-intro indole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino- nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4- nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethyl inosine, 2'0-methoxyethyl nebularine, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4- nitro-benzimidazole, 2'-0-methoxyethyl 3-nitropyrrole, and combinations thereof.

3. The oligonucleotide according to claim 2, wherein the universal base is deoxyinosine, 1- (2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole.

4. The oligonucleotide according to claim 3, wherein the universal base is deoxyinosine.

5. The oligonucleotide according to claim 1, wherein the separation portion comprises contiguous nucleotides having universal bases.

6. The oligonucleotide according to claim 1, wherein the 5 '-high T_m specificity portion is longer than the 3 '-low T_n, specificity portion.

7. The oligonucleotide according to claim 1, wherein the 5'-high T_m specificity portion is 15- 40 nucleotides in length.

8. The oligonucleotide according to claim 1, wherein the 3'-low T_m specificity portion is 3-15 nucleotides in length.

9. The oligonucleotide according to claim 1, wherein the separation portion is 3-10 nucleotides in length.

10. The oligonucleotide according to claim 1, wherein the T_n, of the 5'-high T_m specificity portion ranges from 40°C to 80⁰C.

11. The oligonucleotide according to claim 1, wherein the T_n, of the 3 '-low T_n, specificity portion ranges from 1O⁰C to 40⁰C.

12. The oligonucleotide according to claim 1, wherein the T_mof the separation portion ranges from 3°C to 15⁰C.

13. The oligonucleotide according to claim 1, wherein the separation portion hybridizes with a site showing genetic diversity in the target sequence of the viral genome.

14. The oligonucleotide according to claim 1, wherein the 5'-high T_m specificity portion and the 3 '-low T_n, specificity separation portion hybridize with a conserved sequence in the target sequence of the viral genome.

15. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-26.

16. An oligonucleotide pair hybridizable specifically with a nuclei acid of a respiratory virus, wherein each oligonucleotide 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 N0s: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 sequences of SEQ ID NOs:23 and 24; or each of the nucleotide sequences of SEQ ID NOs:25 and 26.

17. A kit for detecting a respiratory virus, comprising the oligonucleotide of any one of claims 1-15, or the oligonucleotide pair of claim 16.

18. A kit for amplifying a target sequence in a genome sequence of a respiratory virus, comprising the oligonucleotide of any one of claims 1-15, or the oligonucleotide pair of claim 16.

19. A method for detecting a respiratory virus, which comprises hybridizing the oligonucleotide of any one of claims 1-15 with a nucleic acid molecule in a respiratory viral genome.

20. A method for amplifying a nucleic acid molecule of a respiratory virus, which comprises the steps of:

(a) annealing the oligonucleotide of any one of claims 1-15 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.