[go: up one dir, main page]

WO1999053096A2 - Acides nucleiques specifiques du serotype du poliovirus et procedes servant a detecter des serotypes specifiques du poliovirus - Google Patents

Acides nucleiques specifiques du serotype du poliovirus et procedes servant a detecter des serotypes specifiques du poliovirus Download PDF

Info

Publication number
WO1999053096A2
WO1999053096A2 PCT/US1999/007506 US9907506W WO9953096A2 WO 1999053096 A2 WO1999053096 A2 WO 1999053096A2 US 9907506 W US9907506 W US 9907506W WO 9953096 A2 WO9953096 A2 WO 9953096A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
poliovirus
serotype
nucleotide sequence
set forth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/007506
Other languages
English (en)
Other versions
WO1999053096A3 (fr
Inventor
David R. Kilpatrick
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
Original Assignee
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Priority to AU34733/99A priority Critical patent/AU3473399A/en
Publication of WO1999053096A2 publication Critical patent/WO1999053096A2/fr
Publication of WO1999053096A3 publication Critical patent/WO1999053096A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

  • POLIOVIRUS SEROTYPE-SPECIFIC NUCLEIC ACIDS AND METHODS FOR DETECTING SPECIFIC SEROTYPES OF POLIOVIRUS
  • the present invention relates to reagents and methods for the detection of polioviruses.
  • the present invention relates to nucleic acids which may be employed in amplification reactions to selectively detect in a clinical sample vaccine-related or wildtype polioviruses of a particular serotype. while not detecting polioviruses of either of the other two serotypes. and to methods for detecting vaccine-related or wildtype poliovirus of a particular serotype in amplification reactions.
  • enteroviruses are among the most common viral pathogens of humans. These include polioviruses, coxsackieviruses, echoviruses, and the more recently numbered enteroviruses.
  • Poliovirus an RNA virus
  • Poliovirus is the etiologic agent of paralytic poliomyelitis. It is separable, on the basis of specificity of neutralizing antibody, into three serotypes. designated types 1, 2 and 3.
  • type 1 poliovirus has been responsible for about 85 percent of all paralytic poliomyelitis and for most epidemics
  • type 2 poliovirus has been responsible for about 5 percent of paralytic poliomyelitis
  • type 3 poliovirus has been responsible for about 10 percent of paralytic poliomyelitis and for occasional epidemics.
  • the complete nucleotide sequences of all three serotype genomes. and their antigenic structure, are described in H. Toyoda et al..
  • Poliomyelitis worldwide by the year 2000. Constant virologic surveillance for a period of several years after such eradication will be critical to ensure that the virus has in fact been eradicated. All suspected cases of poliomyelitis (clinically diagnosed acute flaccid paralysis) will have to be analyzed for the presence of poliovirus. If poliovirus is found, the serotype of the particular poliovirus isolate will have to be determined (serotype 1, 2 or 3) to confirm clinical diagnosis. The serotype of the isolate will also be imperative for the assessment of the vaccination program in the region where the poliovirus is found in order to further the eradication efforts. Poliovirus genomes evolve rapidly during replication in humans.
  • polioviruses undergo frequent recombination (genetic exchange) in nature.
  • the nucleotide sequences of polioviruses currentl) in circulation throughout the world are extremely heterogenous, as is described by Kew et al.. "Molecular Epidemiology of Wild Poliovirus Transmission,” in Kurstak et al, Applied Virology Research. Vol. 2, 199-221 (Plenum Press, New York, 1990).
  • a typical rate for the fixation of mutations over the entire genome is one to two nucleotide substitutions per week. Although there may be a high degree of conservation at the amino acid level, there is considerable nucleotide variation.
  • Monoclonal Antibodies in Type 1 Poliovirus can be Located Outside or Inside the Antibody Binding Site," J. Virol. 57, 81-90 (1986) and by Weigers and Dernick. "Molecular Basis of Antigenic Structures of Poliovirus: Implications for their Evolution During Morphogenesis, J. Virol. 66, 4597-4600 (1992). Two independent approaches are currently employed for routine identification of polioviruses: (1 ) antigenic characterizations using cross- adsorbed antisera; and (2) molecular characterizations using genotype-specific probes or polymerase chain reaction primers.
  • Serologic methods can generally differentiate vaccine-related isolates from wild polioviruses. However, serologic methods have a limited capacity to differentiate among wild poliovirus genotypes. Also, the sensitivin of polymerase chain reaction in detecting polioviruses is several orders of magnitude greater when compared to serologic methods.
  • Vaccine-related polioviruses may be identified directly using Sabin-strain-specific nucleic acid probes and polymerase chain reaction primers. Wild polioviruses may be identified indirectly by their non-reactivity with the Sabin-strain-specific molecular reagents. Direct identification of wild polioviruses is possible with wild-genotype-specific probes and polymerase chain reaction primer sets. However, the current catalog of wild-genotype-specific molecular reagents does not cover all of the many different poliovirus genotypes still in circulation worldwide. Consequently, identification of wild polioviruses through the exclusive use of Sabin-strain-specific molecular reagents is dependent upon the accurate typing of virus isolates. In many cases, a clinical sample may contain a mixture of polioviruses having different serotypes, and/or polioviruses and nonpolio enteroviruses and/or polioviruses and other viruses.
  • the molecular reagents currently in use do not allow for the rapid or accurate differentiation between the three different serotypes of all polioviruses (vaccine-related or wildtype polioviruses).
  • the standard method for serotyping polioviruses (virus growth in tissue culture and subsequent virus neutralization using serotype-specific antisera) is very time-consuming (i.e., at least one to two weeks) and laborious, and has a rather low sensitivity level compared to molecular based methods.
  • Genomic sequences which encode intervals of strong amino acid conservation can still be highly degenerate. Many different synonymous codon combinations could potentially occur within primer binding sequences. If all degenerate codon combinations were permitted within the target sequences of a set of polymerase chain reaction primers, the primers would have to match an extremeh large number of different sequence combinations.
  • Such reagents and methods will allow the clinician to improve the speed and accuracy of processing large numbers of clinical samples, and to increase the sensitivity of detecting minorit> populations of polioviruses in mixed-serotype cultures. Such reagents and methods will also aid the clinician in patient management, eliminate unnecessar)' tests, improve the speed and accuracy of diagnosis and prognosis, help control poliovirus infection and reduce the use of unnecessary antibiotics. Further, such reagents and methods will aid in the worldwide endeavor to eradicate poliomyelitis.
  • the present invention provides nucleic acids which can be used as primers in pairs in reverse transcription/polymerase chain reactions to rapidly (within approximately 6 hours) detect and distinguish between the three poliovirus serotypes of virtually all genotypes of both wildtype and vaccine-related polioviruses, while not recognizing the other two poliovirus serotypes, nonpoliovirus enteroviruses, or other viruses, with a high degree of specificity and sensitivity.
  • the nucleic acids are specific for one of the three serotypes of both wildtype and vaccine-related polioviruses and, thus, exclude from detection the other two serotypes of poliovirus, and other viruses.
  • the nucleic acids and methods of detection of the present invention permit the direct, rapid, efficient, and accurate determination of whether a vaccine-related or wildtype poliovirus is present in a clinical sample and, if so, the identification of the particular serotype of poliovirus which is present in the sample by amplification in highly-specific reverse transcription' polymerase chain reactions.
  • the serotype of all wild and vaccine-related polioviruses can be specifically identified using one of the serotype-specific primer sets described herein. These primer sets contain mixed bases or deoxyinosine residues at positions of codon degeneracy, and have been found to allow the serotype-specific identification of 213 poliovirus isolates using reverse polymerase/transcription chain reaction.
  • a polymerase chain reaction primer set containing mixed-base and deoxyinosine residues is set forth in D. Kilpatrick et al., "Group Specific Identification of Polioviruses by PCR using Primers Containing Mixed-Base or Deoxyinosine Residues at Positions of Codon Degeneracy," Journal of Clinical Microbiology, 2990-2996 (1996).
  • This polymerase chain reaction primer set cannot distinguish between the three different serotypes of poliovirus. Rather, this polymerase chain reaction primer set appears only to differentiate polioviruses from nonpolio enteroviruses. Further, this primer set has nucleotide sequences which are different from the nucleic acids of the present invention.
  • the present invention provides purified nucleic acids which will selectiveh hybridize with nucleic acids present in one of the three serotypes of virtualh all vaccine-related and wildtype polioviruses, and will not hybridize with ( 1 ) nucleic acids present in either of the other two serotypes of polioviruses. (2) nucleic acids present in nonpolio enteroviruses or (3) nucleic acids present in other viruses.
  • nucleic acids may be used as primers in nucleic acid amplification reactions, such as reverse transcription polymerase chain reactions, to specifically detect only one of the three serotypes of virtualh' all vaccine-related and wildtype polioviruses in a biological sample containing nucleic acids, while not detecting the two other serotypes of vaccine-related or wildtype polioviruses, nonpolio enteroviruses or other types of viruses.
  • nucleic acids are set forth in the Sequence Listing as SEQ ID NOS:l-57.
  • the present invention also provides purified nucleic acids which are substantially the same as the above-described nucleic acids.
  • These nucleic acids ma ⁇ van from the above-described nucleic acids bv one or more nucleotide substitutions, additions and/or deletions, or by the addition of an advantageous feature therein, such as a radiolabel or other label for nucleic acid detection or immobilization, so long as they retain the ability of the above- described nucleic acids to specifically detect only one of the three serotypes of polioviruses and, thus, to distinguish between the three serotypes of polioviruses.
  • the present invention also provides a method for detecting the presence or absence of a specific serotype of poliovirus in a sample containing nucleic acids which comprises amplifying the nucleic acids present in the sample with a primer pair comprising nucleic acids within the present invention, and determining the presence or absence of an amplification product having a size which is characteristic for a serotype 1, 2 or 3 poliovirus, thereby determining the presence or absence of the specific serotype of poliovirus in the sample.
  • the present invention still further provides a kit for determining the presence or absence of a specific serotype of a vaccine-related or wildtype poliovirus in a biological sample containing nucleic acids comprising primers comprised of nucleic acids within the present invention.
  • FIG. 1 is a map of the poliovirus genome and a table of amino acid sequences present in the capsid protein VP1 region of twenty different poliovirus isolates, and shows the conservation of serotype-specific amino acid epitopes. Degenerate PCR primers were designed for these conserved amino acids. Upper area: Untranslated regions are shown as lines in the upper area of FIG. 1 , and the region of the translated polyprotein is represented by the rectangle. Shaded areas indicate locations of surface loops forming neutralization antigenic sites 1 , 2a and 3. Arrows show locations and polarities of the primers of the present invention. Lower Area: Alignment of amino acid residues (bold) whose codons are specifically bound by the primers of the present invention (bold).
  • FIG. 2 is a table which shows the alignment of the primers whose nucleotide sequences are set forth in the Sequences Listing as SEQ ID NOS: 1 -5 with poliovirus target sequences.
  • the targeted amino acid sequences top are aligned with all possible encoding nucleotide sequences (middle) and the sequences of the primers whose nucleotide sequences are set forth in the Sequence Listing as SEQ ID NOS: 1-5 (bottom).
  • Abbreviations for nucleotides follow International Union of Biochemistry nomenclature ("Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences.” Eur. J. Biochem. 150: 1-5 (1985)): H. adenine.
  • cytosine, or thymine cytosine, or thymine
  • I deoxyinosine: N, adenine. cytosine. guanine, or thymine
  • R adenine or guanine
  • Y cytosine or thymine.
  • FIG. 3 shows an ethidium bromide-stained 12% polyacrylamide gel of amplification products produced in the Example described hereinbelow using the serotype 1 -specific primers SeroPVl,2S and SeroPVlA.
  • Lane M is the Boehringer-Mannheim molecular weight marker V, which ranges from 57 bp to 587 bp.
  • Lane 1 contains a Sabin serotype 1 -specific positive control.
  • Lane N contains a negative template control.
  • Lanes 2 through 13 show amplification products obtained with templates of different wild poliovirus isolates: (1 ) PV 1 (panel A): 2. 6070/CHN94; 3. 5558/NIE94; 4, 5386/ANG94; 5. 5058/THA93: 6.
  • FIG. 3A shows serotype 1 wild poliovirus isolates
  • FIG. 3B shows serotype 2 wild poliovirus isolates
  • FIG. 3C shows serotype 3 wild poliovirus isolates.
  • FIGS. 3A, 3B and 3C show that all serotype 1 wild poliovirus isolates yielded a 70 base pair (bp) reverse transcription/polymerase chain reaction product from the poliovirus genome, while none of the serotype 2 or serotype 3 wild poliovirus isolates yielded a 70 bp reverse polymerase/ transcription chain reaction product from the poliovirus genome.
  • FIG. 4 shows an ethidium bromide-stained 12% polyacrylamide gel of amplification products produced in the Example described hereinbelow using the serotype 2-specif ⁇ c primers SeroPVl ,2S and SeroPV2A.
  • Lane M is as described above for FIG. 3.
  • Lane 1 contains a Sabin serotype 2-specif ⁇ c positive control.
  • Lane N contains a negative template control.
  • Lanes 2 through 13 contain the same wild poliovirus isolates as are described above for FIG. 3.
  • FIG 4A shows serotype 1 wild poliovirus isolates;
  • FIG. 4B shows serotype 2 wild poliovirus isolates; and
  • FIG. 4C shows serotype 3 wild poliovirus isolates.
  • FIGs. 4A shows serotype 1 wild poliovirus isolates;
  • FIG. 4B shows serotype 2 wild poliovirus isolates;
  • FIG. 4C shows serotype 3 wild poliovirus isolates.
  • 4B and 4C show that all serotype 2 wild poliovirus isolates yielded a 79 bp reverse transcription/polymerase chain reaction product from the poliovirus genome, while none of the serotype 1 or serotype 3 wild poliovirus isolates yielded a 79 bp reverse transcription/polymerase chain reaction product from the poliovirus genome.
  • FIG. 5 shows an ethidium bromide-stained 12% polyacrylamide gel of amplification products produced in the Example described hereinbelow using the serotype 3-specific primers SeroPV3S and SeroPV3A.
  • Lane M is as described above for FIG. 3.
  • Lane 1 contains the Sabin serotype 3-specific positive control.
  • Lane N contains a negative template control.
  • Lanes 2 through 13 contain the same wild poliovirus isolates as are described above for FIG. 3.
  • FIG. 5A shows serotype 1 wild poliovirus isolates;
  • FIG. 5B shows serotype 2 wild poliovirus isolates; and
  • FIG. 5C shows serotype 3 wild poliovirus isolates.
  • FIGs. 5A shows serotype 1 wild poliovirus isolates;
  • FIG. 5B shows serotype 2 wild poliovirus isolates;
  • FIG. 5C shows serotype 3 wild poliovirus isolates.
  • FIGs. 5A shows serotype 1 wild polio
  • 5B and 5C show that all serotype 3 wild poliovirus isolates yielded a 140 bp reverse transcription/polymerase chain reaction product from the poliovirus genome, while none of the serotype 1 or serotype 2 wild poliovirus isolates yielded a 140 bp reverse transcription/polymerase chain reaction product from the poliovirus genome.
  • nucleic acid which is both the same length as. and exactly complementary in base pairing to, the given nucleic acid.
  • hybridization refers to the formation of a duplex structure two single-stranded nucleic acids due to fully (100%) or less than fully (less than 100%) complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between less than fully complementary nucleic acid strands which contain regions of mismatch due to one or more nucleotide substitutions, deletions or additions. Depending on the length of a primer of the present invention, the primer can generalh range from between about 80% complementary bases and full complementarity with a target region of a nucleic acid and still hybridize therewith under stringent conditions.
  • the degree of complementarity between a primer of the present invention and a target sequence present in a particular serotype of poliovirus must be at least enough to exclude hybridization with nucleic acid present in another serotype of poliovirus, with nucleic acid present in nonpolio enteroviruses and with nucleic acid present in other viruses.
  • the majority of poliovirus serotypes match their respective selective primer of the invention at 100% of the nucleotides (i.e., all of the nucleotides present in the selective primer hybridize to the specific poliovirus template). This is because the degenerate primers, as well as the multiple number of primer species, have the ability to compensate for different codon usages, as long as the amino acid being coded for is the same. More than 90% of the vaccine-related or wildtype poliovirus isolates tested in the Example and sequenced had 100% homology to the polymerase chain reaction primers of the present invention.
  • nucleotides follow the nomenclature described by the Nomenclature Committee for the International Union of Biochemistry. "Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences/' Eur. J. Biochem. 150:1-5 (1985), in which "A” represents adenine residues. "C” represents cytosine residues. "T” represents thymine residues, “G” represents guanine residues, “I” represents deoxyinosine residues, “M” represents adenine or cytosine residues. "R” represents adenine or guanine residues and "Y” represents cytosine or thymine residues.
  • sequences present in a particular serotype of vaccine-related or wildtype poliovirus but does not hybridize with nucleotide sequences present in other serotypes of poliovirus.
  • the primers of the present invention whose nucleotide sequences are set forth herein selectively hybridize with nucleotide sequences present in a particular serotype of vaccine-related or wildtype poliovirus.
  • nucleic acids which are substantially the same as (as defined hereinbelow) these primers will also selectively hybridize with nucleotide sequences present in a particular serotype of vaccine-related or wildtype poliovirus.
  • nucleic acid and oligonucleotide refer to primers, to probes and to oligomer fragments to be detected, and are generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose). to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • nucleic acid and oligonucleotide are used interchangeably herein. These terms refer only to the primary structure of the molecule.
  • Nucleic acids and oligonucleotides can be prepared by any of several well-known methods. For example, they may be prepared by cloning and restriction of desired sequences, or by direct chemical synthesis by the phosphotriester methods described by Narang et al., Meth. Enzymol. 68:90-99 (1979) and Brown et al., Meth. Enzymol. 68:109-151 (1979); by the diethylphosphoramidite method described by Beaucage et al., Tetrahedron Lett. 22:1859-1862 (1981); or by the solid support method described in U.S. Patent No. 4.458.066. A review of nucleic acid syntheses methods is provided in Goodchild. Bioconjugate Chemistry 1(3):165-187 (1990).
  • primer refers to an oligonucleotide. whether natural or synthetic, which is capable of hybridizing with a template nucleic acid (i.e., the nucleic acid being amplified), and which is capable of initiating the synthesis of a DNA extension product having a nucleotide sequence which is complementary to the template nucleic acid strand in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase or reverse transcriptase) present in an appropriate buffer and at a suitable temperature.
  • the length of primers typically ranges between about 10 and about 100 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Primers need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template.
  • Extensions which are not capable of hybridizing with the target nucleic acid may generally be added to primers to allow the performance of a variety of postamplification operations on the amplification product without significant perturbation of the amplification itself.
  • a primer can incorporate an additional feature, such as a radio- or non-radioactive label (biotin. etc.) which will allow for the detection or immobilization of the amplification product, but which will not alter the basic property of the primer (that of acting as a point of initiation of DNA synthesis).
  • a primer may contain an additional nucleic acid sequence at the 5 ' end which will not hybridize to the target nucleic acid, but which will facilitate the cloning of the amplified nucleic acid product.
  • a primer is "specific" for a target sequence if the primer will hybridize with the target sequence, and will not hybridize with non-target sequences.
  • purified means that the nucleic acids are of sufficient purity so that they may be employed, and will function properly, in the methods of the present invention, as well as in a clinical, diagnostic, experimental or other procedure, such as reverse transcription/polymerase chain reaction. Southern or dot blot hybridization or gel electrophoresis. Many procedures are known by those of ordinary skill in the art for purifying nucleic acids prior to their use in other procedures.
  • nucleic acid having a nucleotide sequence which is similar to the nucleotide sequence of one of the nucleic acids set forth in the Sequence Listing as SEQ ID NOS:l-57, and which retains the functions of such nucleic acid, but which differs from such nucleic acid by the substitution, deletion and/or addition of one or more hybridizing or non-hybridizing nucleotides, and/or by the incorporation of some other feature into the nucleic acid, such as a radiolabel or other label (biotin, etc.) for nucleic acid detection or immobilization.
  • a radiolabel or other label biotin, etc.
  • nucleic acids will have the ability of the primers whose nucleotide sequences are set forth in the Sequence Listing to selectively detect in a biological sample during amplification reactions, such as reverse transcription/polymerase chain reaction, nucleic acid present in vaccine-related or wildtype polioviruses of a particular serotype, while not detecting nucleic acid present in polioviruses of the other two serotypes. nucleic acid present in nonpolio enteroviruses. or nucleic acid present in other viruses. Modifications at the 5 ' - end of a nucleic acid can include, for example, the addition of an isotope, such as 32 P, or a chemical, such as digoxigenin.
  • restriction enzyme sites and/or cloning sites can be added to the 5 " - end of a nucleic acid (from about 6 to more than about 12 nucleotides) for the direct cloning of the amplified product.
  • target region and "target nucleic acid” refer to a region of nucleic acid which is to be amplified, detected, or otherwise analyzed.
  • sequence to which a primer hybridizes is referred to as a "target sequence.”
  • the present invention provides purified nucleic acids which will selectively hybridize with nucleic acids present in one of the three serotypes of virtually all vaccine-related and wildtype polioviruses. and will not hybridize with nucleic acids present in either of the other two serotypes of polioviruses, with nucleic acids present in nonpolio enteroviruses or with nucleic acids present in other viruses.
  • nucleic acids allow a clinician to rapidly and accurately determine by amplification reactions, such as reverse transcription/polymerase chain reaction, whether or not poliovirus is present in a biological sample and, if so, the particular serotype of poliovirus which may be present in a sample containing one or more vaccine-related or wildtype polioviruses.
  • amplification reactions such as reverse transcription/polymerase chain reaction
  • nucleic acids of the present invention include DNA primers having the nucleotide sequences set forth below, and/or in the Sequence Listing (SEQ ID NOS: 1-57):
  • SeroPV1.2S (2459-2477) 1 or 2 SEQ ID NO 1 5'-TGCGIGA(T/C)ACIACICA(T/C)AT-3'
  • SeroPVIA (2528-2509) 1 SEQ ID NO 2 5'-ATCATICT(T/C)TCIA(A/G)CAT(T/C)TG-3'
  • SeroPV2A 2537-2518 2 SEQ ID NO 3 5'-A(T/C)ICC(T/C)TClACI(A/G)CICC(T/C)TC-3'
  • SeroPV3S (3037-3056) 3 SEQ ID NO 4 5'-AA(C/T)CCITCI(A/G)TITT(C/T)TA(C/T)AC-3
  • SeroPV3A (3176-3157) 3 SEQ ID NO:5 5'-CCIAl(T/C)TGITC(A/G)TTIG(T/C)(A/G)TC-3
  • a cDNA prepared from these RNA nucleotide sequence intervals by reverse transcription will bind to the indicated DNA molecules (primers) by complementary base pairing. Deoxyinosine residues are indicated by the letter I. Primer positions having equimolar amounts of two different nucleotides are enclosed in parenthesis. Generally, these primers are intended to be used in primer sets for the identification of specific poliovirus serotypes. For example, primers SeroPVl ,2S and SeroPVlA are used together as a primer set in an amplification reaction to specifically identify serotype 1 polioviruses.
  • primers SeroPV1.2S and SeroPV2A are used together as a primer set in an amplification reaction to specifically identify serotype 2 polioviruses
  • primers SeroPV3S and SeroPV3A are used together as a primer set in an amplification reaction to specifically identify serotype 3 polioviruses.
  • These pairs of primers are degenerate panpoliovirus (panPV) primer sets which can be used to distinguish between the three serotypes of vaccine-related and wildtype polioviruses in clinical samples. These primer sets will not detect the other two serotypes of polioviruses, and will not detect other viruses to any significant extent.
  • primers were targeted near poliovirus gene VP1 sequences encoding a domain thought to interact with cell receptors.
  • the consensus nucleotide sequence which is the degenerate primer containing both deoxyinosine and mixed-base nucleotide positions, set forth in SEQ ID NO: l for primer SeroPV1.2S denotes the four possible combinations (species) of nucleotides that are found in SEQ ID NOS:6-9. as set forth below:
  • SEQ ID NO:2 for primer SeroPVlA denotes the eight possible combinations (species) of nucleotides that are found in SEQ ID NOS: 10-17, as set forth below: SEQ ID NUMBER NUCLEIC ACID SEQUENCE
  • the consensus nucleotide sequence set forth in SEQ ID NO:3 for primer SeroPV2A denote the sixteen possible combinations (species) of nucleotides that are found in SEQ ID NOS:18-33. as set forth below:
  • SEQ ID NO:4 for primer SeroPV3S denotes the eight possible combinations (species) of nucleotides that are found in SEQ ID NOS:34-41 , as set forth below: SEQ ID NUMBER NUCLEIC ACID SEQUENCE
  • the consensus nucleotide sequence set forth in SEQ ID NO:5 for primer SeroPV3A denotes the sixteen possible combinations (species) of nucleotides that are found in SEQ ID NOS:42-57. as set forth below:
  • the canyon structure on the poliovirus surface that is postulated to bind to cell receptors is primarily formed from conserved intervals of the capsid proteins VPl and VP2.
  • the complete poliovirus capsid protein VPl alignments which are present in a poliovirus amino acid sequence database located at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, revealed several areas which contained amino acid sequences which were unique to a particular poliovirus serotype. This is shown in FIG. 1. The greatest serotype- specific amino acid sequence conservation was found near the 5 ' -end of the poliovirus VPl gene.
  • the three sets of degenerate, serotype-specific polymerase chain reaction primers for poliovirus differentiation described hereinabove were then designed to identify (i.e.. be fully complementary to) nucleotide intervals in the poliovirus VPl gene which encode the amino acid epitopes described above.
  • degenerate codon positions on the poliovirus genome template were matched by mixed bases (i.e., more than one base used at a particular nucleotide position) or by deoxyinosine residues on the polymerase chain reaction primers. This was done even though some investigators have reported unsatisfactory losses in polymerase chain reaction sensitivity and diagnostic specificity when using degenerate primers. Because deoxyinosine residues can pair with all four of the nucleotide bases, deoxyinosine residues were used in those positions where 3 or 4 different nucleotides were possible.
  • primers SeroPVl,2S (SEQ ID NOT), SeroPVlA (SEQ ID NO:2), SeroPV2A (SEQ ID NO:3).
  • SeroPV3S SEQ ID NO:4
  • SeroPV3A SEQ ID NO:5
  • Serotype 1 primer SeroPVlA was designed to recognize the nucleotide sequence encoding the amino acid sequence QMLESMI (SEQ ID NO:58).
  • Serotype 2 primer SeroPV2A was designed to recognize the nucleotide sequence encoding the amino acid sequence EGVVEGV (SEQ ID NO:59).
  • Serotype 3 primer SeroPV3A was designed to recognize the nucleotide sequence encoding the amino acid sequence DANDQIG (SEQ ID NO:60).
  • the nucleotide sequence of serotype 3 primer SeroPV3A is complementary to a nucleotide sequence located in a region of the poliovirus VPl gene which has been identified as poliovirus neutralization site 2a. as is described in P. Minor et al.. "Antigenic Structure of Polioviruses of Serotypes 1, 2 and 3.” J Gen. Virol 67:1283-1291 (1986).
  • the 5 " -end of the poliovirus VPl gene also contains a conserved amino acid sequence for serotype 3 poliovirus isolates (amino acid numbers 9 through 15 of the poliovirus capsid protein VPl)
  • the primer initialh' designed for the nucleotide sequence which encodes this conserved amino acid sequence had higher levels of degeneracy at its 3'-end.
  • experiments performed with this primer to detect a large collection of wild serotype 3 polioviruses produced inconsistent amplification results. These inconsistent amplification results were believed to be due to the presence of deoxyinosine residues near the 3 ' -end of the primer, which may result in a lower discrimination between bases, as is discussed in M.
  • the appropriate upstream conserved amino acid sequences were also identified, and included (FIG. 1): (1) the amino acid sequence LRDTTHI (SEQ ID NO:61 ) for poliovirus serotypes 1 and 2 (amino acids numbers 3225 through 3231 of the poliovirus capsid protein VPl); and (2) the amino acid sequence NPSIFYT (SEQ ID NO:62) for poliovirus serotype 3 (amino acids numbers 1 178 through 1 184 of the poliovirus capsid protein VPl). These upstream amino acid sequences are present in all known polioviruses, and in a few closely-related picornaviruses.
  • Serotype 1 and 2 primer SeroPVl ,2S was designed to recognize the nucleotide sequence encoding the amino acid sequence LRDTTHI
  • serotype 3 primer SeroPV3S was designed to recognize the nucleotide sequence encoding the amino acid sequence NPSIFYT.
  • the serotype-specific antisense polymerase chain reaction primers (SeroPVlA, SeroPV2A, and SeroPV3A) were determined to recognize nucleotide sequences encoding amino acid sequences which are only found in polioviruses. Thus, these primers do not amplify nucleic acids present in non-poliovirus enteroviruses or in other viruses. This is an important characteristic of these primers because, in many cases, polioviruses and non-poliovirus enteroviruses and/or other viruses may be present in the same clinical sample.
  • the serotype-specific degenerate polymerase chain reaction primers set forth hereinabove are believed to be the only known polymerase chain reaction primers which can be employed in amplification reactions to specifically detect one of the three poliovirus serotypes in both vaccine-related and wildtype polioviruses. while not detecting the other two serotypes of polioviruses, and not detecting nonpolio enteroviruses or other viruses.
  • nucleic acids within the scope of the invention include, but are not limited to, the nucleic acids described herein.
  • Contemplated equivalents of the nucleic acids described herein include nucleic acids which otherwise correspond thereto, and which have the same general properties thereof, wherein one or more simple variations are made which do not adversely affect the function of the nucleic acids as described herein.
  • nucleic acids of the present invention include
  • DNA molecules which are substantially the same as the nucleic acids having the nucleotide sequences set forth below, and in the Sequence Listing as SEQ ID NOST-57.
  • Modifications to the nucleic acids set forth in the Sequence Listing as SEQ ID NOS: 1-57 can be made so long as they do not prevent these nucleic acids from annealing to cDNAs prepared from the conserved target polioviral sequences from which they were derived.
  • modified nucleic acids are within the scope of the present invention if they have the ability to function as primers in amplification reactions to specifically detect, in a biological sample, vaccine-related or wildtype polioviruses of one serotype, while not detecting polioviruses of either of the other two serotypes, nonpolio enteroviruses or other viruses.
  • Computer programs are readily available to the skilled artisan which can be used to compare modified nucleotide sequences to previously published nucleotide sequences of polioviruses to select the most appropriate sequences for amplification. The specificity of these sequences for the different poliovirus serotypes can be determined by conducting a computerized comparison with known sequences catalogued in GENBANK. a computerized database, using the computer programs Word Search of FASTA of the Genetics Computer Group (Madison. WI). which search the catalogued nucleotide sequences for similarities to the nucleic acid in question.
  • the modified nucleic acids of the invention have at least 80% homology (and preferably at least 85%, 90%, 95%, 97%, 98%, or 99% homology) with the nucleotide sequences set forth in the Sequence Listing as SEQ ID NOS: l - 57, or at least 80% complementarity (and preferably at least 85%, 90%, 95%, 97%. 98%, or 99% complementarity) with the nucleic acid sequences to which nucleic acids having the nucleotide sequence set forth in the Sequence Listing as SEQ ID NOS: 1 -57 hybridize.
  • the nucleic acids of the present invention can be used as primers in amplification reactions to specifically detect one of the three serotypes of vaccine- related or wildtype poliovirus in a clinical sample, without detecting the other two serotypes of poliovirus. nonpolio enteroviruses or other viruses.
  • these nucleic acids may be used as primers to amplify nucleic acid which is unique to individual vaccine-related or wildtype poliovirus serotypes in accordance with the methods of the present invention.
  • the nucleic acids of the present invention can be used as primers in reverse transcription of viral RNA from poliovirus isolates, and can vary in length. These oligonucleotides are typically between about 10 and about 100 nucleotides in length, preferabh' between about 12 and about 30 nucleotides in length, and most preferably between about 15 and about 25 nucleotides in length. There is no standard length for optimal polymerase chain reaction amplification or reverse transcription. An optimal length for a particular application is readily determined in the manner described in H. Erlich, PCR Technology. Principles and Application for DNA Amplification. (1989). Several computer software programs are available to facilitate primer design, for example. T.
  • nucleic acids identified by SEQ ID NOS:l and 2, 1 and 3, and 4 and 5, respectively selectively hybridize with nucleic acid present in a poliovirus of serotype 1, serotype 2, and serotype 3, respectively.
  • Nucleic acids present in a particular serotype of vaccine-related or wildtype poliovirus can, thus, be detected with the nucleic acids of the present invention utilizing a nucleic acid amplification technique, such as reverse transcription/polymerase chain reaction as taught in the Example described hereinbelow.
  • serotype-specific polymerase chain reaction primers which hybridize only with nucleic acids containing a target sequence from a particular serotype of vaccine-related or wildtype poliovirus are utilized.
  • the absence of such an amplification product after the performance of the amplification technique indicates that the particular serotype of poliovirus is not present in the sample.
  • Amplification products may be separated, for example, by electrophoresis on polyacrylamide gels, and visualized with ethidium bromide staining.
  • the degenerate serotype-specific polymerase chain reaction primers of the present invention can be utilized in polymerase chain reactions in the combinations (with different species) of nucleotide sequences set forth in the Sequence Listing as SEQ ID NOS: 6-9 (for Primer SeroPVl,2S); SEQ ID NOST0-17 (for Primer SeroPVlA); SEQ ID NOS: 18-33 (for Primer SeroPV2A); SEQ ID NOS:34-41 (for Primer SeroPV3S): and SEQ ID NOS:42-57 (for Primer SeroPV3A).
  • the nucleic acids of the present invention can be utilized in any of a number of nucleic acid detection techniques including, but not limited to, reverse transcription/polymerase chain reaction, isothermal DNA amplification, liquid hybridization, etc.
  • nucleic acids of the present invention can be labeled or tagged for use in radioactive, chemiluminescence, fluorescent, or other detection systems.
  • nucleic acids of the present invention may be prepared and tested for the ability to selectively hybridize with a target nucleic acid in the manner described herein, or by modifications thereof, using readily-available starting materials, reagents and equipment.
  • a preferred method for preparing and testing these nucleic acids is described hereinbelow in the Example.
  • the present invention provides a method for detecting the presence or absence of a serotype 1 vaccine-related or wildtype poliovirus in a sample containing nucleic acids, said method comprising the steps of :
  • the present invention provides a method for detecting the presence or absence of a serotype 2 vaccine-related or wildtype poliovirus in a sample containing nucleic acids, said method comprising the steps of:
  • the present invention provides a method for detecting the presence or absence of a serotype 3 vaccine-related or wildtype poliovirus in a sample containing nucleic acids, said method comprising the steps of:
  • the primers employed in this amplification reaction are a first primer having the nucleotide sequence set forth in the Sequence Listing as SEQ ID N0:4 and a second primer having the nucleotide sequence set forth in tn the Sequence Listing as SEQ ID NO:5, the amplification product will have approximately 140 bp if the serotype 3 poliovirus is present.
  • the present invention provides a method for detecting the presence or absence of serotype 1. serotype 2. and/or serotype 3 vaccine-related or wildtype poliovirus in a sample suspected of containing one or more serotypes of vaccine-related or wildtype polioviruses. said method comprising the steps of:
  • step (d) determining the presence or absence of an amplification product for each of steps (a), (b) and (c). whereby the presence of an amplification product having a size which is characteristic for serotype 1 poliovirus resulting from step (a) indicates the presence of serotype 1 vaccine-related or wildtype poliovirus in the sample, the presence of an amplification product having a size which is characteristic for serotype 2 poliovirus resulting from step (b) indicates the presence of serotype 2 vaccine-related or wildtype poliovirus in the sample, and the presence of an amplification product having a size which is characteristic for serotype 3 poliovirus resulting from step (c) indicates the presence of serotype 3 vaccine- related or wildtype poliovirus in the sample.
  • the invention provides a method for detecting the presence or absence of a particular serotype of vaccine-related or wildtype poliovirus in a sample containing nucleic acids upon the amplification of a target nucleotide sequence by an in vitro reverse transcription/polymerase chain reaction.
  • the polymerase chain reaction for amplifying DNA
  • the reverse transcription polymerase chain reaction for amplifying cDNA generated from RNA. as would be used in amplification reactions performed with an RNA virus
  • These methods may be used for the rapid detection of viruses from clinical samples, such as feces. nasal wash, rectal swab, cerebrospinal fluid, throat swab, lung biopsy tissue and like materials.
  • These specimens ma ⁇ be collected by methods known in the art. such as by the methods described in T. Chonmaitree et al.. "Comparison of Cell Cultures for Rapid Isolation of Enteroviruses/' J. Clin. Microbiol. 26:2576-2580 ( 1988). and in C. Hall. "Clinicalh Useful Method for the Isolation of Respiratory Syncytial Virus.” J Infect. Dis. 131 : 1-5 (1975).
  • the nucleic acids present in a sample which are being amplified ma ⁇ be a single- or double-stranded DNA or RNA. If the starting material is RNA. such as in polioviruses. reverse transcriptase is used to prepare a first strand cDNA prior to conventional polymerase chain reaction.
  • RNA such as in polioviruses.
  • reverse transcriptase is used to prepare a first strand cDNA prior to conventional polymerase chain reaction.
  • General information concerning polymerase chain reaction, and the amplification of specific sequences of nucleic acids is present in U.S. Patent No. 4,683.195; U.S. Patent No. 4.683.202; U.S. Patent No. 4.965,188; U.S. Patent No. 5.578.467; U.S. Patent No. 5.545.522; U.S. Patent No. 5.624.833: F. M.
  • a double-stranded target nucleic acid sequence present in a sample is denatured and. due to the presence of a large molar excess of the primers, primers are annealed to each strand of the denatured target sequence.
  • the primers oriented with their 3 ' ends pointing towards each other, hybridize to opposite strands of the target sequence and. due to the action of DNA polymerase. prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates.
  • the two primers anneal to opposite ends of the target nucleic acid sequence, and in orientations such that the extension product of each primer is a complementary copy of the target nucleic acid sequence and.
  • the primers of the present invention are complementary to a cDNA generated b ⁇ reverse transcription of a serotype-specific poliovirus degenerate nucleotide sequence
  • Denaturation of nucleic acid strands usually takes place at about 94°C
  • the normal annealing (55 to 60°C) and extension (65 to 72°C) temperatures generalh used for in vitro amplification b ⁇ reverse transcription/poly merase chain reaction are generalh unsuitable for use with the degenerate primers of the piesent ⁇ n ⁇ ention because the presence of deoxy inosine residues results in low annealing temperatures with man ⁇ pohourus cDNA templates
  • the optimal annealing temperatuie was determined to be approximatel ⁇ 42°C.
  • the annealing temperature which ma ⁇ be employ ed with the primers and methods of the present invention ranges from about 38°C to about 46°C. and is preferably about 42°C
  • the extension temperature was decreased to 60°C.
  • the extension temperature which may be employed with the primers and methods of the present inv ention ranges from about 56°C to about 64°C, and is preferably about 60°C When these preferred annealing and extension conditions were used in the Example described hereinbelow. sequences of all poliov irus cDNA templates tested were efficiently amplified
  • reaction times are from about 15 seconds to about 1 mmute denaturing, from about 30 seconds to about 1 5 mmutes of annealing, and from about 30 seconds to about 1 5 minutes of extension
  • the optimum time for each of these three reactions is 45 seconds pei reaction
  • Suitable assay formats for detecting amplification products or hybrids formed between probes and target nucleic acid sequences in a sample are described, for example, in F. M. Ausubel et al., "Current Protocols in Molecular Biology.” Greene Publishing Associates and Wiley-Interscience. (John Wiley and Sons. New York ( 1987; updated quarterly)); Sambrook et al.. Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y. ( 1985). Examples of these assay formats include the dot-blot and reverse dot-blot assay formats.
  • amplified target DNA is immobilized on a solid support, such as a nylon membrane.
  • a solid support such as a nylon membrane.
  • the membrane-target complex is incubated with labeled probe under suitable hybridization conditions, unhybridized probe is removed by washing under suitable stringent conditions, and the membrane is monitored for the presence of bound probe.
  • An alternate format is a "reverse" dot-blot format, in which the amplified target DNA is labeled and the probes are immobilized on a solid support, such as a nylon membrane.
  • the target DNA is typically labeled during amplification by the incorporation of labeled primers therein.
  • One or both of the primers can be labeled.
  • the membrane-probe complex is incubated with the labeled amplified target DNA under suitable hybridization conditions, unhy bridized target DNA is removed by washing under suitably stringent conditions, and the filter is then monitored for the presence of bound target DNA.
  • the present invention also provides a kit for detecting a nucleic acid present in a particular serotype of vaccine-related or wildtype poliovirus by nucleic acid amplification comprising one of the primer pairs described hereinabove.
  • the kit will contain the three primer pairs specifically described in the Example, and instructions describing the use of these three primer sets in the detection of a specific serotype of poliovirus (the suggested buffer, primer concentrations, and the like).
  • the following Example describes and illustrates the methods for the preparation of serotype-specific primers within the present invention, and the methods for using these primers in reverse transcription/polymerase chain reactions to rapidly and accurately detect one of the three serotypes of poliov iruses in a sample, while not detecting either of the other two serotypes of polioviruses.
  • This Example is intended to be merely illustrative of the present invention, and not limiting thereof in either scope of spirit. Those of skill in the art will readily understand that variations of the conditions and processes of the procedures described in this Example can be used to prepare and test these primers.
  • the 52 v accine-related and 161 wildtype poliov irus isolates set forth in Tables 1 and 2 were emplo ⁇ ed in the amplification reactions (reverse transcription poly merase chain reactions) to test the ability of degenerate polymerase chain reaction primer pairs within the present invention to selecti ely detect a particular serotype of poliovirus. while not detecting the other two seroty pes ol poliov irus These poliov irus isolates had been prev iously characterized as being of serotype 1. serotype 2. or serotype 3 by neutralization with hype ⁇ mmune equine sera, partial genomic sequencing and probe hy bridization as described by Kew et al .
  • SeroPVlA SEQ ID NO:2
  • SeroPV2A SEQ ID NO:3
  • SeroPV3S SEQ ID NO:4
  • SeroPV3A SEQ ID NO:5
  • the primers were prepared by the ⁇ - cyanoethyl phosphoramidite method using an automated synthesizer (Model 380A. Applied Biosystems, Foster City, CA), as is described by Sinha et al., "Polymer Support Oligonucleotide Synthesis XVIII; use of ⁇ -cyanoethyl-N.N-dialkylamino- N-morpholino phosphoramidite of Deoxynucleosides for the Synthesis of DNA fragments Simplifying Deprotection and Isolation of the Final Product," Nucleic Acids Research 12. 4539-4557 (1984).
  • the primer pairs used in the amplification reactions were: (1) SeroP ⁇ T .2S (SEQ ID NOT ) and SeroPVlA (SEQ ID NO:2) to detect nucleic acid present in serotype 1 polioviruses; (2) SeroPV1.2S (SEQ ID NOT ) and SeroPV2A (SEQ ID NO:3) to detect nucleic acid present in serotype 2 polioviruses: and (3) SeroPV3S (SEQ ID NO:4) and SeroPV3A (SEQ ID NO:5) to detect nucleic acid present in serotype 3 polioviruses.
  • Primer nucleotide positions indicated by a mixed base pair used equal amounts of the two different nucleotides indicated.
  • dCTP, dGTP. and dTTP Pulacia. Piscataway. NJ
  • 5 U of placental ribonuclease inhibitor Boehringer Mannheim Biochemicals. Indianapolis. IN.
  • AMV avian myeloblastosis virus
  • Taq DNA polymerase Perkin Elmer-Cetus, Norwalk. CT.
  • Three separate reverse transcription/polymerase chain reactions were performed for each of the viral isolates described in Table 1 or Table 2.
  • AMY reverse transcriptase. and Taq DNA polymerase were overlaid with mineral oil. heated for 5 minutes at 95°C to release virion RNA and chilled on ice. The enzymes were then added, and the samples were incubated at 42°C for 30 minutes. followed by 30 cycles of programmed amplification (denaturation: 94°C. 1 minute: annealing: 42°C. 1 minute: extension: 60°C, 1 minute) in a DNA thermal cycler (Perkin Elmer-Cetus).
  • Table 1 lists the 52 vaccine-related and 161 wildtype poliovirus isolates tested with each of the above-described degenerate primer pairs in these reverse transcription/polymerase chain reactions.
  • These poliovirus isolates represent all major poliovirus genotypes presently known in nature, and include wildtype and vaccine-related poliovirus isolates of each of the three poliovirus serotypes from virtually every geographic region of the world. All of these isolates were tested in amplification reactions containing RNAs obtained from these isolates with each of the serotype-specific polymerase chain reaction primer pairs set forth hereinabove. The serotypes of all of the 213 poliovirus isolates tested were correctly identified using these serotype-specific polymerase chain reaction primer sets.
  • RNAs of all poliovirus isolates tested served as efficient templates for amplification in reverse transcription/polymerase chain reaction assays using the primer sets described hereinabove.
  • AU serotype 1 isolates listed in Table 1 and Table 2. which were amplified with the serotype 1 -specific primer pair SeroP ⁇ T .2S and SeroPV lA yielded a 70 bp amplification- product.
  • FIG. 3A shows the specific amplification of 12 of the wildtype serotype 1 polioviruses listed in Table 2. which is typical of all of the serotype 1 polioviruses tested, by reverse transcription/polymerase chain reaction using this primer set.
  • FIGs. 3B and 3C respectively, no amplification products of the characteristic size (70 bp) were seen when this primer pair was tested with isolates representing wild poliovirus serotypes 2 or 3. All serotype 2 isolates listed in Table 1 and Table 2 which were amplified with the serotype 2-specific primer pair SeroPVl ,2S and SeroPV2A yielded a 79 bp amplification product.
  • FIG. 4B shows the specific amplification of 12 of the wildtype serotype 2 polioviruses listed in Table 2.
  • FIG. 5C shows the specific amplification of 12 of the wildtype serotype 3 polioviruses listed in Table 2, which is typical of all of the serotype 3 polioviruses tested, by reverse transcription/polymerase chain reaction using this primer set. Sources for the products in each lane are described in the BRIEF DESCRIPTION OF THE DRAWINGS. Reverse transcription, polymerase chain reaction analysis of serotype 1 isolates (FIG. 5A) and serotype 2 isolates (FIG.
  • the level of sensitivity for detecting polioviruses from tissue culture isolates using the above amplification conditions was found to be similar between degenerate and non-degenerate poly merase chain reaction primers This sensitiv ity , with a v irus detection limit of approximately 200 pfu.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des acides nucléiques qui effectueront une hybridation sélective avec l'acide nucléique présent dans virtuellement la totalité des poliovirus associés à un vaccin ou des poliovirus de type sauvage d'un sérotype déterminé, mais n'effectueront aucune hybridation avec des acides nucléiques présents dans des poliovirus de l'un ou l'autre des deux autres sérotypes, des acides nucléiques présents dans des entérovirus autres que poliovirus ou des acides nucléiques présents dans d'autres virus. On peut utiliser ces acides nucléiques en tant qu'amorces dans des réactions de polymérase en chaîne ou de transcription inverse afin de détecter de façon spécifique la présence d'un des trois sérotypes de poliovirus associés à un vaccin ou de type sauvage dans un spécimen clinique contenant des acides nucléiques. Ces acides nucléiques ne détecteront pas la présence des deux autres sérotypes de poliovirus ou la présence d'entérovirus autres que poliovirus ou la présence d'autres virus dans le spécimen. Elle concerne également des procédés servant à détecter de façon spécifique la présence d'un des trois sérotypes de poliovirus dans un spécimen clinique contenant des acides nucléiques au moyen des acides nucléiques que l'invention concerne, dans une réaction de transcription inverse ou de polymérase en chaîne in vitro.
PCT/US1999/007506 1998-04-15 1999-04-06 Acides nucleiques specifiques du serotype du poliovirus et procedes servant a detecter des serotypes specifiques du poliovirus Ceased WO1999053096A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU34733/99A AU3473399A (en) 1998-04-15 1999-04-06 Poliovirus serotype-specific nucleic acids and methods for detecting specific serotypes of poliovirus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US8194598P 1998-04-15 1998-04-15
US60/081,945 1998-04-15

Publications (2)

Publication Number Publication Date
WO1999053096A2 true WO1999053096A2 (fr) 1999-10-21
WO1999053096A3 WO1999053096A3 (fr) 1999-11-18

Family

ID=22167411

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/007506 Ceased WO1999053096A2 (fr) 1998-04-15 1999-04-06 Acides nucleiques specifiques du serotype du poliovirus et procedes servant a detecter des serotypes specifiques du poliovirus

Country Status (2)

Country Link
AU (1) AU3473399A (fr)
WO (1) WO1999053096A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013095119A1 (fr) * 2011-12-14 2013-06-27 De Staat Der Nederlanden, Vert. Door De Minister Van Vws Identification de souches de poliovirus
CN116904448A (zh) * 2022-05-13 2023-10-20 北京中科生仪科技有限公司 一种脊髓灰质炎检测引物探针组合物及一体化微流控芯片试剂盒

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585477A (en) * 1993-07-13 1996-12-17 Us Health Poliovirus specific primers

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013095119A1 (fr) * 2011-12-14 2013-06-27 De Staat Der Nederlanden, Vert. Door De Minister Van Vws Identification de souches de poliovirus
CN116904448A (zh) * 2022-05-13 2023-10-20 北京中科生仪科技有限公司 一种脊髓灰质炎检测引物探针组合物及一体化微流控芯片试剂盒

Also Published As

Publication number Publication date
AU3473399A (en) 1999-11-01
WO1999053096A3 (fr) 1999-11-18

Similar Documents

Publication Publication Date Title
Chen-Fu et al. Detection and identification of vaccine-related polioviruses by the polymerase chain reaction
Kilpatrick et al. Group-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residue at positions of codon degeneracy
Thoelen et al. Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000
AU693465B2 (en) Poliovirus specific primers and methods of detection utilizing the same
US20100279273A1 (en) Nucleic acid sequences for the amplification and detection of respiratory viruses
Kämmerer et al. Nested PCR for specific detection and rapid identification of human picornaviruses
Zell et al. Detection of porcine enteroviruses by nRT–PCR: differentiation of CPE groups I–III with specific primer sets
Kilpatrick et al. Serotype-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy
De et al. Identification of vaccine-related polioviruses by hybridization with specific RNA probes
CA2365870C (fr) Typage d'enterovirus humains
US6168917B1 (en) Detection and identification of non-polio enteroviruses
Gjøen et al. Specific detection of Coxsackie viruses A by the polymerase chain reaction
Chezzi Rapid diagnosis of poliovirus infection by PCR amplification
US20020142293A1 (en) Enteroviral polynucleotides, methods of detecting enteroviruses and kits containing the polynucleotides
Schweiger et al. Differentiation of vaccine and wild-type polioviruses using polymerase chain reaction and restriction enzyme analysis
WO1999053096A2 (fr) Acides nucleiques specifiques du serotype du poliovirus et procedes servant a detecter des serotypes specifiques du poliovirus
AU1219301A (en) Serotype-specific identification of enterovirus 71 by RT-PCR
Takeda et al. Differentiation between wild and vaccine-derived strains of poliovirus by stringent microplate hybridization of PCR products
Nel et al. A nucleotide-specific polymerase chain reaction assay to differentiate rabies virus biotypes in South Africa
Chapman et al. Identification of coxsackie B viruses and enteroviruses in general using nucleic acid hybridization
AU2005201742B2 (en) Detection and identification of enteroviruses
CA2503468C (fr) Detection et identification d'enterovirus
Mixed-Base Serotype-Specific Identification of Polioviruses
CA2600979A1 (fr) Procede et matieres associees

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase