[go: up one dir, main page]

WO2001034848A2 - Identification specifique au serotype de l'enterovirus 71 par rt-pcr - Google Patents

Identification specifique au serotype de l'enterovirus 71 par rt-pcr Download PDF

Info

Publication number
WO2001034848A2
WO2001034848A2 PCT/US2000/029021 US0029021W WO0134848A2 WO 2001034848 A2 WO2001034848 A2 WO 2001034848A2 US 0029021 W US0029021 W US 0029021W WO 0134848 A2 WO0134848 A2 WO 0134848A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nucleic acid
nucleotide sequence
primer
same
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/US2000/029021
Other languages
English (en)
Other versions
WO2001034848A3 (fr
Inventor
Betty Ann Brown
David R. Kilpatrick
Mark A. Pallansch
M. Steven Oberste
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 AU12193/01A priority Critical patent/AU1219301A/en
Priority to EP00973713A priority patent/EP1230399A2/fr
Priority to CA002391362A priority patent/CA2391362A1/fr
Publication of WO2001034848A2 publication Critical patent/WO2001034848A2/fr
Publication of WO2001034848A3 publication Critical patent/WO2001034848A3/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

  • the present invention relates to reagents and methods for the detection and analysis of enterovirus 71
  • the present invention relates to nucleic acids, as well as kits and methods comprising these nucleic acids for detecting, amplifying, and sequencing nucleic acids of enterovirus 71
  • CA 16 and EV71 are closely related genetically, but EV71 is also associated with severe neurologic disease such as encephalitis, meningitis, cranial nerve palsies, Guillan-Barre syndrome, and poliomyelitis-like syndrome much more frequently than is CA16 (reviewed in Alexander, J P , et al , 'Enterovirus 71 infections and neurologic disease - United States, 1977- 1991 " J Infect Dis 169; 905-908 (1994), Chumakov, M , et al , "Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria," Arch Virol , 60 329-340 (1979), Gilbert et al , Outbreak of enterovirus
  • Enterovirus 71 exhibits a wide variation in clinical presentation EV71 has been associated with severe central nervous system disease with a case- fatality rate of 0% to 6% (Landry et al , 1995)
  • case- fatality rate 0% to 6%
  • Enterovirus 71 exhibits a wide variation in clinical presentation EV71 has been associated with severe central nervous system disease with a case- fatality rate of 0% to 6% (Landry et al , 1995)
  • a large EV71 outbreak in Bulgaria in 1975 (705 reported cases) there were 149 cases of paralytic disease and 44 fatalities Forty-five cases of EV71 infection were reported in the United States in 1987, including eight cases of paralysis and one fatality (Alexander J P , et al , 1994), and virus circulation was widespread, with isolates reported in at least seventeen states
  • enterovirus serotypes are defined by neutralization using immune sera directed against the capsid proteins, nucleotide and ammo acid sequences of the capsid region correlate with serotype (Oberste et al ,
  • the present invention provides nucleic acids which can be used as primers in amplification and sequencing reactions to rapidly (generally within approximately 6 hours) amplify and sequence EV71 nucleic acids
  • a preferred group of nucleic acids of the present invention when used as primer pairs in amplification reactions, detect EV71 with a high degree of specificity and sensitivity With these preferred primer pairs, the specificity of amplification
  • Polymerase chain reaction primer sets containing mixed-base and, in some cases, deoxyinosine residues is set forth in D Kilpat ⁇ ck, "Poliovirus specific primers and methods of detection utilizing the same," U S Pat. No 5,691 134
  • the disclosed polymerase chain reaction primer sets distinguish between the three different serotypes of poliovirus and differentiate polioviruses from nonpolio enteroviruses Amplification reactions utilizing the disclosed primers do not amplify EV71 nucleic acid Further, the primer sets have nucleotide sequences which are different from those of the nucleic acids of the present invention
  • a polymerase chain reaction primer set containing mixed-base and deoxyinosine residues is set forth in D Kilpat ⁇ ck et al (1996) This polymerase chain reaction primer set appears to differentiate polioviruses from nonpolio enteroviruses Amplification reactions utilizing the disclosed primers do not amplify EV71 nucleic acid Further, this primer set has nucleotide sequences which are different from those of the nucleic acids of the present invention None of the above-described publications teaches or describes the primers, or methods and kits using these primers
  • the present invention provides nucleic acids which can be used as primers in amplification and sequencing reactions to rapidly amplify and sequence target EV71 nucleic acids
  • nucleic acid primers are set forth in the Sequence Listing as SEQ ID NOS 1-12 A preferred group
  • nucleic acids of the present invention when used as primer pairs in amplification reactions, detect EV71 with a high degree of specificity and sensitivity.
  • the specificity of amplification methods of the present invention is such that target EV71 nucleic acids are amplified to a detectable level, whereas no detectable product is obtained when CA16 nucleic acids are used.
  • the present invention also provides purified nucleic acids which are complementary to nucleic acids having a nucleotide sequence selected from SEQ ID NOS 1-12.
  • the present invention also provides purified nucleic acids which are substantially the same as the above-described nucleic acids.
  • These nucleic acids may vary from the above-described nucleic acids by one or more nucleotide substitutions, additions and/or deletions, or by the addition of an advantageous feature therein, such as, for example, a radiolabel or other label for nucleic acid detection or immobilization, so long as they retain the ability of the above-described nucleic acids.
  • the present invention also provides a method for detecting the presence or absence of EV71 in a sample containing nucleic acids, including clinical samples.
  • the method 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 EV71 , thereby determining the presence or absence of EV71 in the sample.
  • the present invention still further provides a kit for determining the presence or absence of EV71 in a biological sample.
  • the kit contains the nucleic acid primers disclosed in the present invention.
  • Figure 1 is a dendrogram generated by neighbor-joining method with the DNADIST distance measure (Phylip 3.5). The phylogram was calculated based on the nucleotide divergence of the VP1 gene (position 2442 to 3332). The last four or five characters of strain name indicate the state or country and two digit year of isolation. Branch lengths are proportional to the number of nucleotide differences and the frequencies with which the branches for genotype A, B, and C appeared in 1000 bootstrap replications (i e 898, 543 and 999 respectively) Clades with bootstrap numbers are expressed in percentile The marker bar denotes a measurement of the relative phylogenetic distance (The branch length for the outgroup CA16 was reduced by 0 75 for space consideration )
  • Figure 2 is an alignment of genotype-consensus VP1 ammo acid sequences
  • the EV71 consensus sequence shows ammo acid residues that are identical in at least 85% of all strains (upper case letters) and those that are identical in at least 50%, but less than 85% of all strains (lower case letters) Sites that are identical in all strains of all genotypes are double- underlined, those that are identical in all strains of genotypes B and C, but different in BrCr-CA-70, are single-underlined
  • the genotype consensus sequences indicate sites of at least 85% consensus among all strains of a given genotype (hyphens) and sites that are characteristic of one or more genotypes (upper case, 85% consensus within genotype, lower case 50% to 85% consensus within genotype)
  • Figure 3 illustrates the position of PCR primers relative to the ammo acid sequences of VP3 and VP1 (Brown et al , 'Complete nucleotide sequence of Enterovirus 71 is distinct from poliovirus," Virus Res , 39 195-205 (1995), Poyry et al , "Molecular analysis of coxsackievirus A16 reveals a new genetic group of enteroviruses, ' Virology, 292 982-987 (1994)) Primer position and sense are indicated by arrows Ammo acids at the annealing sites for primers 159S and 162A are boxed Ammo acids at the annealing sites for primers 92S and 93A are underlined Dots indicate sequences not shown Numbers above sequence indicate relative ammo acid positions in VP3 and VP1
  • Figure 4 shows ethidium bromide-stained gel sections containing amplification products produced by RT-PCR of EV71 RNA in the Examples described hereinbelow using the primer pair 159S/162A or the primer pair 92S/93A
  • Sources of templates for the products in each lane were as follows (A) (Primer pair 159S/ 162A and EV71 genotype A and genotype B strains), 1 , CA70-BrCr, 2, NY72-2228, 3, AUS74-2610 4, MN78-10181 , 5, CA79- 2258 6 TN80-21 14 7 OH82-2381 , 8, OK87-6910, 9 AL88-8149, 10 MS87- 7423, (B) (Primer pair 159S/ 162A and EV71 genotype C strains), 1 , AK87- 7238 2, MA87-0915, 3 TX89-9978, 4, TX91 -0443 5, NC94-1997, 6 VA95- 2132 7 AUS95-2640 8 MA97
  • nucleic acid and oligonucleotide refer to primers, probes, and/or oligomer fragments to be detected, and are generic to polydeoxy ⁇ bonucleotides (containing 2-deoxy-D- ⁇ bose), to poly ⁇ bonucleotides (containing D- ⁇ bose), and to any other type of polynucleotide which is an N glycoside of a pu ⁇ ne or py ⁇ midine base, or modified pu ⁇ ne or py ⁇ midine base.
  • nucleic acids and oligonucleotides are used interchangeably herein These terms refer only to the primary structure of the molecule Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA 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 phosphot
  • 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 (at least 85%, preferably at least 90%, and more preferably at least 95% complimentary) to hybridize with the template.
  • 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.
  • the phrase 'selectively hybridize indicates that the stringency of hybridization conditions can be set such that a nucleic acid hybridizes with target nucleic acid sequences present in EV71 , but not with nucleotide sequences present in CA16
  • the primers 93A and 162A of the present invention selectively hybridize with target EV71 nucleic acids This is demonstrated by the results of Example 2 which indicate that either of these primers, in combination with another primer that does not selectively hybridize, can be used to amplify EV71 target nucleic acids under conditions wherein CA16 nucleic acids are not amplified
  • hybridization refers to the formation of a duplex structure by 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
  • 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 transc ⁇ ption/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 1-77, 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 ⁇ on-hyb ⁇ dizmg nucleotides and/or by the incorporation of some other feature into the nucleic acid, such as a radiolabel or other label (biotm etc ) for nucleic acid detection or immobilization
  • These nucleic acids will have the ability of the primers whose nucleotide sequences are set forth in the Sequence Listing to detect in a biological sample during amplification reactions such as reverse transc ⁇ ption/polymerase chain reaction nucleic acids present in EV71 Modifications at the 5'- end of a nucleic acid can include for example, the addition of
  • primer pair' refers to two primers that each hybridize to different target sequences on different strands of a DNA molecule to prime amplification of a target nucleic acid
  • the primers are oriented upon hybridization with the target nucleic acid with their 3' ends pointing towards each other and prime enzymatic extension along the nucleic acid target in the presence of the four deoxy ⁇ bonucleotide t ⁇ phosphates
  • the primers of a primer pair when used in an amplification reaction are capable of amplifying a target nucleic acid located between the primers
  • target region and “target nucleic acid” refer to a region of nucleic acid that is to be amplified detected sequenced, or otherwise analyzed
  • target sequence ' refers to the sequence to which a primer hybridizes
  • nucleic acid sequences and the relative genomic position of the "VP1 "and VP3" genes are disclosed in GenBank entries referenced in Brown and Pallansch, 1995 and Poyry et al , 1994
  • amplification reaction mixture refers to a mixture comprising four different nucleoside t ⁇ phosphates and an agent for polymerization, preferably DNA polymerase or reverse transcriptase, present in an appropriate buffer Oligonucleotide Primers Used for RT-PCR and Sequencing of Target EV71 Nucleic Acids
  • the present invention provides purified nucleic acids which hybridize with nucleic acids present in the EV71 VP1 gene and which function as primers for amplification and sequencing of target nucleic acids of the EV71 VP1 gene
  • primers preferably contain sites of mixed-base composition and deoxyinosine at sites of fourfold codon degeneracy
  • EV71 target nucleic acids are amplified for further analysis, such as for nucleic acid sequence analysis
  • 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 , 2, and 5-53)
  • sense-oriented pr ⁇ mers such as 159S, 161 S, and the like
  • antisense primers such as 174A, 198A, and the like
  • the following are effective primer pairs for amplification and sequencing 159S/162A, 159S/204A, 161 S/NP1A, 169S/NP1A, 163S/ 174A, 172S/174A, and 197S/198A
  • the consensus nucleotide sequence (SEQ ID NO 1 ) is the degenerate primer containing mixed-base nucleotide positions for primer 159S and denotes the four possible combinations (species) of nucleotides that are found in SEQ ID NOS 5-8, as set forth below
  • the consensus nucleotide sequence set forth in SEQ ID NO 2 for primer 162A denotes the sixteen possible combinations (species) of nucleotides that are found in SEQ ID NOS 18-33, as set forth below
  • the consensus nucleotide sequence set forth in SEQ ID NO:8 for primer 161 denotes the four possible combinations (species) of nucleotides that are found in SEQ ID NOS:34-37, as set forth below:
  • the consensus nucleotide sequence set forth in SEQ ID NO:10 for p ⁇ mer 163 denotes 8 possible combinations (species) of nucleotides that are found in SEQ ID NOS:38-45, as set forth below:
  • the consensus nucleotide sequence set forth in SEQ ID NO:11 for primer 169 denotes the four possible combinations (species) of nucleotides that are found in SEQ ID NOS:46-49, as set forth below:
  • SEQ ID NO: 13 The consensus nucleotide sequence set forth in SEQ ID NO: 13 for primer NP1A denotes the four possible combinations (species) of nucleotides that are found in SEQ ID NOS:50-53, as set forth below:
  • preferred nucleic acids which form primer pairs wherein one of the primers of the primer pairs selectively hybridizes to EV71 nucleic acids
  • primer pairs When these primer pairs are used in an amplification reaction, EV71 target nucleic acids are amplified, but not CA16 nucleic acids Determination of the desired nucleotide sequence for these EV71 serotype-specific primers is facilitated, in part, by data obtained using the oligonucleotide primers used for RT-PCR and sequencing of EV71 nucleic acid described above
  • Pairs of EV71 -specific PCR primers can be designed using conserved motifs which amplify many strains of EV71 In a preferred embodiment, these pairs of primers are designed so that a target nucleic acid of EV71 is amplified, but no amplification product is detectable for CA16 nucleic acid
  • at least one primer of the primer pair selectively hybridizes to a region of VP1
  • one primer of the primer pair hybridizes to sequences at the carboxyl-termmus of the VP3 gene and the other primer of the primer pair selectively hybridizes near the center of VP1 gene (Fig 3)
  • primers preferably contain sites of mixed- base composition and deoxyinosine at sites of fourfold codon degeneracy
  • 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 1-4, and 54-77)
  • primers 159S and 162A are preferably used together as a primer set in an amplification reaction to specifically identify EV71
  • primers 92S and 93A are preferably used together as a primer set in an amplification reaction to specifically identify EV71
  • SEQ ID NO 3 The consensus nucleotide sequence set forth in SEQ ID NO 3 for primer 92S denote the sixteen possible combinations (species) of nucleotides that are found in SEQ ID NOS 54-69, as set forth below
  • SEQ ID NO 69 b'- I I GAG I I I I I I ACI I AC A I G The consensus nucleotide sequence set forth in SEQ ID NO 4 for primer 93A denotes the eight possible combinations (species) of nucleotides that are found in SEQ ID NOS 70-77 as set forth below
  • degenerate codon positions on the EV71 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 The use of deoxyinosine residues in primers is discussed in F Martin et al , "Base Pairing involving Deoxyinosine Implications from Probe Design," Nucleic Acids Res , 13, 8927-8938 (1985), in M Batzer et al , "Enhanced Evolutionary PCR
  • primers constructed to hybridize in the VP1 gene region of EV71 can be successfully used to amplify and sequence this region in a given EV71 isolate
  • the current invention facilitates the construction of other primers for amplifying EV71 target nucleic acids by providing fourteen examples of nucleotide sequences that are effective for this amplification
  • the current invention facilitates the construction of other primers for amplifying target nucleic acids in the EV71 genome by providing the sequence of the VP1 gene for 113 strains of EV71 (available in GenBank sequence database, accession numbers AF009522 to AF009559 and AF135867 to AF135949, AF135911 , AF135935, and AF135941 to AF135950) Therefore primers can be constructed from highly conserved nucleotides across many strain
  • 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 NOS 1-77
  • Modifications to the nucleic acids set forth as SEQ ID NOS 1-77 can be made so long as they do not prevent these nucleic acids from annealing to cDNAs prepared from the conserved target EV71 sequences from which they were derived
  • a radiolabel or non-radiolabel for nucleic acid detection or immobilization can be made so long as they do not prevent these nucleic acids from annealing to cDNAs prepared from the conserved target EV71 sequences from which they were derived
  • oligonucleotide primers used for RT-PCR and sequencing of EV71 nucleic acid described above such modified nucleic acids are within the scope of the present invention if they have the ability to function as primers in amplification or sequencing reactions for EV71 nucleic acids
  • the EV71 serotype-specific primer pairs described above such modified nucleic acids are within the scope of the present invention if they have the ability when used with a second EV71 serotype-specific primer, to detect EV71 but not
  • the modified nucleic acids of the invention have at least 85% homology (and preferably at least 90%, 95% 97%, 98%, or 99% homology) with the nucleotide sequences set forth in the Sequence Listing as SEQ ID NOS 1-77 or at least 85% complementarity (and preferably at least 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 1-77 hybridize
  • the nucleic acids of the present invention can be used as primers in amplification reactions to detect EV71 or as primers in reverse transcription of viral RNA from EV71 isolates
  • These oligonucleotides are typically between about 10 and about 100 nucleotides in length, preferably between about 12 and about 30 nucleotides in length, and most preferably between about 15 and about 25 nucleotides in length
  • An optimal length for a particular application is readily determined in the manner described in H Er ch PCR Technology, Principles and Application for DNA Amplification, (1989)
  • nucleic acids of the invention which hybridize with different regions of nucleic acid present in EV71 should be employed so as to amplify a desired region
  • these two nucleic acids should hybridize to opposite strands of the EV71 DNA in reverse orientation such that they direct extension toward each other Nucleic acids present in EV71 can, thus be detected with the nucleic acids of the present invention utilizing a nucleic acid amplification technique, such as reverse transc ⁇ ption/polymerase chain reaction as taught in the Examples described herembelow
  • EV71 polymerase chain reaction primers which hybridize to EV71 nucleic acids are utilized.
  • the lack of a detectable amplification product after the performance of the amplification technique indicates that EV71 is not present in the sample
  • Amplification products may be separated, for example, by electrophoresis on polyacrylamide or agarose gels, and visualized with ethidium bromide staining
  • the degenerateEV71 polymerase chain reaction primers of the present invention can be utilized in polymerase chain reactions in the combinations of nucleotide sequences set forth in the Sequence Listing as SEQ ID NOS 14-17 (for Primer 159S), SEQ ID NOS 18- 33 (for Primer 162A), SEQ ID NOS 34-37 (for Primer 161), SEQ ID NOS 38- 45 (for Primer 163), SEQ ID NOS 46-49 (for Primer 169), SEQ ID NOS 50-53 (for Primer NP1A), SEQ ID NOS 54-69 (for Primer 92S), and SEQ ID NOS 70-77 (for Primer 93A)
  • 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 transc ⁇ ption/polymerase chain reaction, isothermal DNA amplification, liquid hybridization, etc It is also contemplated that the nucleic acids of the present invention can be labeled or tagged for use in radioactive, chemiluminescence, fluorescent, or other detection systems In general the nucleic acids of the present invention may be prepared and tested for the ability to hybridize with an EV71 target nucleic acid in the manner described herein, or by modifications thereof, using readily-available starting materials, reagents, and equipment However, a preferred method for preparing and testing these nucleic acids is described herembelow in the Examples
  • the present invention provides a method for detecting the presence or absence of EV71 in a sample suspected of containing nucleic acids of enterovirus 71 said method comprising
  • the second primer selectively hybridizes to nucleic acids of enterovirus 71
  • the first primer and the second primer recognize target sequences in an enterovirus 71 region selected from the group consisting of VP1 and VP3, preferably the second primer recognizes a target sequence in the VP1 region of enterovirus 71
  • an enterovirus 71 region selected from the group consisting of VP1 and VP3, preferably the second primer recognizes a target sequence in the VP1 region of enterovirus 71
  • One of ordinary skill in the art preferably with the aid of a computer program, could use the teachings of this disclosure to devise primers that hybridize to the VP1 or the VP3 genes of all known strains of EV71
  • primers that hybridize to EV71 but not CA16 hybridize to a portion of the VP1 gene of EV71
  • the first primer is a purified nucleic acid comprising the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO 1 , or a nucleic acid that is substantially the same as the purified nucleic acid comprising the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO 1
  • the second primer is a purified nucleic acid comprising the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO 2
  • the first primer is a purified nucleic acid comprising the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO 3, or a nucleic acid that is substantially the same as the purified nucleic acid comprising the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO 3
  • the second primer is
  • the amplification method is a reverse transc ⁇ ption/polymerase chain reaction
  • the polymerase chain reaction (for amplifying DNA) and the reverse transcription polymerase chain reaction (for amplifying cDNA generated from RNA, as would be used in amplification reactions performed with an RNA virus) are rapid methods for increasing the copy number of, and sensitively detecting, specific nucleic acid sequences
  • These methods may be used for the rapid detection of viruses from clinical samples, such as feces, nasal wash, rectal swab, cerebrospmal fluid, throat swab, lung biopsy tissue, and like materials
  • These specimens may 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
  • 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 deoxy ⁇ bo ⁇ ucleotide t ⁇ phosphates
  • 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 when separated from its complement, can hybridize to the other primer
  • the end product is then denatured again for another cycle After this three-step cycle has been repeated between about 25 and about 40 times, preferably about 30 times, amplification of a nucleic acid segment by more than one million-
  • the primers of the present invention are complementary to a cDNA generated by reverse transcription of an EV71 nucleotide sequence
  • Denaturation of nucleic acid strands usually takes place at about 94 X
  • the normal annealing (about 55 to about 60X) and extension (about 65 to about 72 X) temperatures generally used for in vitro amplification by reverse transc ⁇ ption/polymerase chain reaction are generally unsuitable for use with the degenerate primers of the present invention because the presence of deoxyinosine residues results in low annealing temperatures with many poliovirus cDNA templates
  • the optimal annealing temperature was determined to be approximately 42 X, near the temperature optimum for avian myeloblastosis virus reverse transcriptase Annealing temperatures above about 46 X or below about 38 X generally reduce the yield of the specific amplicon, and increase the generation of nonspecific amplification products
  • the annealing temperature which may be employed with the primers and methods of the present invention ranges from about 38X to about 46 X, and is preferably about 42X
  • the extension temperature was decreased to 60X which is still within the range for
  • Suitable assay formats for detecting amplification products or hybrids formed between probes and target nucleic acid sequences in a sample are generally well known and are described, for example, in F M Ausubel et al , 1987 Sambrook et al , Molecular Cloninq-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y (1985) Examples of these assay formats include dot-blot and reverse dot-blot assay formats In a dot- blot format amplified target DNA is immobilized on a solid support, such as a nylon membrane The membrane-target complex is incubated with labeled probe under suitable hybridization conditions, unhyb ⁇ dized probe is removed by washing under suitable 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
  • the present invention also provides a kit for detecting enterovirus 71 by nucleic acid amplification comprising one the primer pairs described heremabove
  • the kit will contain one (or more) of the primer pairs specifically described in the Example 2, and instructions describing the use of these primer pairs in the detection of enterovirus 71
  • the kit may also include, for example, suitable buffers, PCR enzymes, standards, and the
  • the 1 13 EV71 strains examined in this study are listed in Table 3, with year and state or country of isolation, and, if known, associated clinical symptoms ("NA” indicates unknown or not reported)
  • the strains were isolated between 1970 and 1998 at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, from 25 different laboratories of state health departments in the United States, and from 5 national enterovirus laboratories in other countries.
  • Viruses were isolated from original clinical specimens by using a variety of cell lines and further propagated in rhabdomyosarcoma cells prior to sequencing. Most isolates were typed by neutralization assay with monospecific rabbit ant ⁇ -EV71 antiserum.
  • Viral RNA was extracted from 200 ⁇ l of cell culture supernatant using UltraSpec III (Biotecx, Houston, Tx.) and resuspended in 20 ⁇ l of water or with the Qiamp Viral RNA kit (Qiagen Inc., Valencia, Calif).
  • the primers used for reverse transcription-polymerase chain reaction (RT-PCR) and sequencing are listed in Table 2. The following were effective primer pairs for amplification and sequencing: 159S/162A; 159S/204A; 161 S/NP1A; 169S/NP1A; 163S/ 174A; 172S/174A; and 197S/198A.
  • the VP1 gene was amplified as a series of overlapping fragments in a one-tube RT-PCR reaction containing 2 ⁇ l of RNA, 20 pmol of each primer, 100 mM each dNTP, 2 mM MgCI 2 , 67 mM T ⁇ s-HCI (pH 8 8), 17 mM (NH 4 ) 2 S0 4 , 1 mM ⁇ -mercaptoethanol, 0 2 mg/ml gelatin, 10 U placental RNase inhibitor (Boeh ⁇ nger Mannheim Biochemicals, Indianapolis, Ind ), 12 U AMV reverse transcriptase (Boeh ⁇ nger Mannheim), and 5 U Taq polymerase (Boeh ⁇ nger Mannheim), in a total volume of 50 ⁇ l
  • the amounts of primer used in each reaction depends on the number of species of that primer Generally, 5 pmoies per species were used in each reaction VP1 -specific cDNA was synthesized by incubation of the reaction mixture for 30 mm at
  • nucleotide sequence data generated in this analysis have been deposited in the GenBank sequence data base, accession numbers AF009522 to AF009559 and AF135867 to AF1 35949, AF13591 1 , AF135935, and AF1 35941 to AF135950.
  • the synonymous substitution rate (Ks) was calculated from the number of nucleotide substitutions per synonymous site by using the computer program Diverge (Genetics Computer Group, “Program Manual for the GCG Package version 9.0” (1996)) based on a method of Li, W. H. et al. "A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes," Mol.
  • VP1 gene sequences 891 nucleotides were determined for 1 13 EV71 strains isolated in the United States, Australia, Colombia, China, Canada, and Malaysia from 1970 to 1998.
  • a phylogenetic tree constructed by the neighbor-joining method indicated that EV71 strains were monophyletic with respect to other enterovirus serotypes ( Figure 1 ).
  • Genotype A contained a single member, BrCr-CA-70, the EV71 prototype, and differed from all other isolates by 16.5% to 19.7 %.
  • Genotype B was represented by 65 strains isolated from 1972 to 1997 in the United States, Australia, Colombia, and Malaysia (Sarawak, island of Borneo).
  • Genotype C represented by 47 strains isolated from 1986 to 1998, included viruses from the United States, Australia, China, Canada, and Malaysia (mainland).
  • Genotypes B and C were further subdivided into clusters within each genotype, two for genotype B ( Figure 1 B) and two for genotype C ( Figure 1 C).
  • Cluster B1 contained isolates from the United States and Australia during the 1970s, as well as a few U.S. isolates from the 1980s (21 14-TN-80, 51 15-TX- 83, 6762-OK-86, and 6910-OK-87). Strains in cluster B1 were more diverse than the B1 strains, differing by up to 8.3% within the cluster and by 6.9% to 1 1.1 % from other genotype B strains.
  • Cluster B2 contained strains isolated in the U.S. from 1981 to 1987, including most isolates from the 1987 nationwide EV71 outbreak.
  • Strain 6658-COL-94 was genetically distinct from all other genotype B strains (5.8% to 1 1.1 % difference), but differed from strains of genotype C by 15.5% to 17.2%.
  • Strain 0731 -MAA-97 a typical representative of many Sarawak, Malaysia strains, was also distinct from other genotype B strains by 6.5% to 10.5% and differed from genotype C strains by 17.1 % to 18.3%.
  • the earliest genotype C strains in our collection were isolated in China in 1985 and Australia in 1986 ( Figure 1 C). Genotype C isolates differed from those of genotype B by 15.5% to 18.7%.
  • Cluster C1 was composed of isolates from the United States and Australia from 1986 to 1995, as well as a 1997 isolate from peninsular Malaysia.
  • Cluster C2 was composed of U.S. and Australian isolates from 1995 to 1998. A 1985 isolate from China appeared to be intermediate between clusters C1 and C2. Viruses in cluster C1 differed from one another by 1.0% to 6.3% and from those in cluster C2 by 6.1 % to 10.1 %, while isolates in cluster C2 differed from one another by 0.7% to 1.1 %. Among all the EV71 isolates, 82% of the predicted VP1 amino acid residues were invariant ( Figure 2). In comparison, the VP1 amino acids of Echovirus 30 isolates are at least 88% identical.
  • the EV71 prototype strain, BrCr-CA-70 (genotype A) was 94.2% to 96.0% identical in VP1 amino acid sequence to all other EV71 isolates.
  • Genotype B isolates were at least 97.9% identical to one another, whereas the genotype C isolates were 98.9% identical to one another.
  • Residues 58, 184, 240 and 289 varied among different genotypes, but were invariant within a genotype. At four other sites (residues 43, 124, 249, and 292), the predominant amino acid at the site differed between genotypes B and C ( Figure 2).
  • R 2 denotes linear regression coefficient 0 25 isolates, 1972 - 1987 c 39 isolates. 1986-1987 d weighted average for sets B1 and C1
  • the 124 EV71 strains examined in this study include the 1 13 strains listed in Table 3 and the following additional isolates, with year and state or country of isolation: OK97-2354, CA87-3105, CA88-3104, CA90- 3103, MD87-9256, and TAI98-2731 , TAI98-2732, TAI98-2733, TAI98-2734, TAI98- 2735, and TAI98-2785.
  • the 125 EV71 strains were isolated between 1970 and 1998 at the Centers for Disease Control and Prevention, Atlanta, Georgia, from 25 different laboratories of state health departments in the United States, and from 5 national enterovirus laboratories in other countries.
  • CA16 strains tested included CA16-G10-51 (prototype), TX88-8799, PA88-8888, IL89-0255, PA89-9544, PA89-9263, PA89-9280, PA89-9281 , PA89-9282, PA89-9283, TN89-9328, TN89-9330, KY89-9352, OR89-9354, OR89-9359, TX89-9538, SD89-9704, MN89-9712, WA89-9840, WA89-9838, WA89-9389, NC89-9853, PA90-9876, NM92-1450, TX92-1576, TX92-1577, TN92-1603, PA94-5753, CT94-2004, CT94-
  • enterovirus prototype strains were tested and included echoviruses 1 to 9, 1 1 to 21 , 24 to 27, and 29 to 33; coxsackievirus A1 to A6, A8 to A22, A24, and B1 to B6; Polio Sabinl , Polio Sabin2, and Polio Sabin3; and enteroviruses 68, 69,70, and 71 .
  • Viruses were isolated from original clinical specimens by using various cell culture lines and further propagated in human rhabdomyosarcoma cells. Most isolates were typed by neutralization assay; all EV71 strains and six (20%) of the CA16 strains were sequenced in the VP1 gene region by dideoxy sequencing.
  • Viral RNA was extracted from 200 ⁇ l of cell culture supernatant using by UltraSpec III (Biotecx) and resuspended in 20 ⁇ l of water or extracted with the Qiamp Viral RNA kit (Qiagen).
  • the primers used for RT-PCR are listed in Table 2.
  • RT-PCR amplifications were performed in a one-tube RT-PCR assay containing 2 ⁇ l of RNA, 20 pmol of each primer, 100 M each dNTP, 2 mM MgCI 2 , 67 mM Tris-HCI (pH 8.8), 17 mM (NH 4 ) 2 S0 4 , 1 mM b- mercaptoethanol, 0.2 mg/ml gelatin, 10 U placental RNase inhibitor (Boehri ⁇ ger Mannheim), 12 U AMV reverse transcriptase (Boehringer Mannheim), and 5 U Taq polymerase (Boehringer Mannheim), in a total volume of 50 ⁇ l.
  • VP1 -specific cDNA was synthesized by incubation of the reaction mixture for 30 min at 42X and 3 min at 94X, and amplified by 30 cycles of 94X for 45 sec, 42X for 45 sec, and 60X for 1 min. Reaction products (10 ⁇ l each) were visualized by ethidium bromide staining and UV transillumination following electrophoretic separation in 12% polyacrylamide gels for 71-bp products or in 1 % agarose gels for 484-bp products.
  • Primers 92S and 93A were directed to sequences encoding the motifs VELFTYM (VP1-123 to VP1-129) and CTPTG(E/Q/R)V (VP1-140 to VP1- 146), respectively ( Figure 3 and Table 2). These primers produced a 71-bp amplification product from EV71 RNA templates derived from 20 geographically and temporally distinct strains ( Figure 4 and Table 2). Primers 92S and 93A did not amplify RNA templates from any of 19 CA16 strains, nor did they amplify templates from the prototype strains of 60 other serotypes (Table 5). Table 5.
  • a second EV71 -specific primer pair, 159S and 162A was designed for use in sequencing and molecular epidemiology studies to provide for the rapid genotyping of EV71 isolates during outbreaks.
  • Primers 159S and 162A were directed to sequences encoding the motifs TMKLCKD (VP3-225 to VP3- 232) and VACTPTG (VP1-138 to VP1-144), respectively ( Figure 3 and Table 2). These primers produced a 484 bp amplification product from EV71 RNA templates derived from 125 geographically and temporally distinct strains ( Figure 4, Table 3, and Table 5.
  • Primers 159S and 162A did not amplify RNA templates from any of 31 CA16 strains (Table 5).

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 pouvant être utilisés comme amorces dans des réactions d'amplification ou de séquençage visant, respectivement, à amplifier ou à séquencer rapidement des acides nucléiques EV71. Un groupe préféré d'acides nucléiques, lorsqu'il est utilisé comme paires d'amorces dans des réactions d'amplification, détecte EV71 avec une spécificité et une sensibilité importantes. Grâce à ces paires préférées d'amorces, la spécificité des procédés d'amplification est telle que l'acide nucléique EV71 est amplifié à un niveau détectable, tandis que l'ADN CA16 ne l'est pas. Les amorces d'acide nucléique de la présente invention contiennent des bases mixtes ou des restes de déoxyinosine à des positions de dégénérescence de codons. Des exemples d'amorces d'acide nucléique pour l'amplification et le séquençage de l'acide EV71 sont illustrés dans la liste des séquences sous SEQ ID NOS : 1-12. Des exemples d'amorces préférées pour la discrimination entre EV71 et CA16 sont illustrés dans la liste des séquences sous SEQ ID NOS : 1-4.
PCT/US2000/029021 1999-11-10 2000-10-20 Identification specifique au serotype de l'enterovirus 71 par rt-pcr Ceased WO2001034848A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU12193/01A AU1219301A (en) 1999-11-10 2000-10-20 Serotype-specific identification of enterovirus 71 by RT-PCR
EP00973713A EP1230399A2 (fr) 1999-11-10 2000-10-20 Identification specifique au serotype de l'enterovirus 71 par rt-pcr
CA002391362A CA2391362A1 (fr) 1999-11-10 2000-10-20 Identification specifique au serotype de l'enterovirus 71 par rt-pcr

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16452099P 1999-11-10 1999-11-10
US60/164,520 1999-11-10

Publications (2)

Publication Number Publication Date
WO2001034848A2 true WO2001034848A2 (fr) 2001-05-17
WO2001034848A3 WO2001034848A3 (fr) 2002-02-07

Family

ID=22594878

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/029021 Ceased WO2001034848A2 (fr) 1999-11-10 2000-10-20 Identification specifique au serotype de l'enterovirus 71 par rt-pcr

Country Status (4)

Country Link
EP (1) EP1230399A2 (fr)
AU (1) AU1219301A (fr)
CA (1) CA2391362A1 (fr)
WO (1) WO2001034848A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014397A1 (fr) * 2001-08-09 2003-02-20 Biomedlab Corporation Sonde destinee a la detection de virus enteriques, kit de detection et procede de detection de virus enteriques
CN101812538B (zh) * 2009-11-13 2012-01-04 镇江市疾病预防控制中心 一种检测肠道病毒71型的荧光定量rt-pcr试剂盒
WO2012060779A1 (fr) * 2010-11-02 2012-05-10 Singapore Polytechnic Méthode de détection de l'entérovirus ev71
CN101812535B (zh) * 2009-02-24 2012-08-22 江苏默乐生物科技有限公司 肠道病毒ev71检测特异性引物和探针
CN114540548A (zh) * 2022-02-28 2022-05-27 贵州安康医学检验中心有限公司 一种基于多交叉恒温扩增和金纳米生物传感器

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520789B (zh) * 2015-09-09 2019-05-10 中国人民解放军军事医学科学院微生物流行病研究所 一种dna分子与重组病毒及它们的制备方法和用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2267648A1 (fr) * 1996-10-02 1998-04-09 David Kilpatrick Detection et identification d'enterovirus non poliovirus
DE60020143T2 (de) * 1999-03-31 2006-02-23 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by the Secretary, Centers for Disease Control and Prevention, Office of Technology Transfer, Atlanta Typisierung von humanen enteroviren

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014397A1 (fr) * 2001-08-09 2003-02-20 Biomedlab Corporation Sonde destinee a la detection de virus enteriques, kit de detection et procede de detection de virus enteriques
CN101812535B (zh) * 2009-02-24 2012-08-22 江苏默乐生物科技有限公司 肠道病毒ev71检测特异性引物和探针
CN101812538B (zh) * 2009-11-13 2012-01-04 镇江市疾病预防控制中心 一种检测肠道病毒71型的荧光定量rt-pcr试剂盒
WO2012060779A1 (fr) * 2010-11-02 2012-05-10 Singapore Polytechnic Méthode de détection de l'entérovirus ev71
CN114540548A (zh) * 2022-02-28 2022-05-27 贵州安康医学检验中心有限公司 一种基于多交叉恒温扩增和金纳米生物传感器

Also Published As

Publication number Publication date
AU1219301A (en) 2001-06-06
WO2001034848A3 (fr) 2002-02-07
CA2391362A1 (fr) 2001-05-17
EP1230399A2 (fr) 2002-08-14

Similar Documents

Publication Publication Date Title
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
Kämmerer et al. Nested PCR for specific detection and rapid identification of human picornaviruses
Le Guyader et al. Evaluation of a degenerate primer for the PCR detection of human caliciviruses
Zell et al. Detection of porcine enteroviruses by nRT–PCR: differentiation of CPE groups I–III with specific primer sets
ES2370169T3 (es) Composiciones y métodos para detectar el virus de la hepatitis b.
AU775950B2 (en) Typing of human enteroviruses
AU693465B2 (en) Poliovirus specific primers and methods of detection utilizing the same
US7714122B2 (en) Nucleic acid molecules and kits including VP1 and VP3 nucleic acid molecules, useful for detecting and identifying enteroviruses
ES2300375T3 (es) Composiciones y metodos para la deteccion del virus de la inmunodeficiencia humana 2 (hiv-2).
US6168917B1 (en) Detection and identification of non-polio enteroviruses
Gjøen et al. Specific detection of Coxsackie viruses A by the polymerase chain reaction
WO2001034848A2 (fr) Identification specifique au serotype de l'enterovirus 71 par rt-pcr
US20020142293A1 (en) Enteroviral polynucleotides, methods of detecting enteroviruses and kits containing the polynucleotides
Tryfonos et al. Molecular typing and epidemiology of enteroviruses in Cyprus, 2003–2007
EP1013775A1 (fr) Réaction de polymèrase en chaine quantitative utilisant un système de détection flurogene en temps réel
WO2003000887A1 (fr) Sonde polynucleotidique et amorce originaire du virus de l'hepatite e des japonais, puces pourvues de cette sonde, kits dotes de cette sonde et procede pour detecter le virus de l'hepatite e au moyen de cette sonde
US20050158710A1 (en) Detection of enterovirus nucleic acid
WO1999053096A2 (fr) Acides nucleiques specifiques du serotype du poliovirus et procedes servant a detecter des serotypes specifiques du poliovirus
Chapman et al. Identification of coxsackie B viruses and enteroviruses in general using nucleic acid hybridization
JPH04504803A (ja) Ｈｈｖ／ｓｉｅゲノムから誘導された核酸断片と診断試薬及びｈｈｖ６感染の診断法
AU2005201742B2 (en) Detection and identification of enteroviruses
CA2503468C (fr) Detection et identification d'enterovirus
Mixed-Base Serotype-Specific Identification of Polioviruses
WO2003091270A1 (fr) Detection du virus mhv

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ 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 MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ 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 MZ SD SL SZ TZ 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
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ 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 MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ 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 MZ SD SL SZ TZ 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

WWE Wipo information: entry into national phase

Ref document number: 2391362

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2000973713

Country of ref document: EP

Ref document number: 12193/01

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 2000973713

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2000973713

Country of ref document: EP