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WO1997039010A1 - Probes for the detection of human papillomavirus - Google Patents

Probes for the detection of human papillomavirus Download PDF

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Publication number
WO1997039010A1
WO1997039010A1 PCT/US1997/006354 US9706354W WO9739010A1 WO 1997039010 A1 WO1997039010 A1 WO 1997039010A1 US 9706354 W US9706354 W US 9706354W WO 9739010 A1 WO9739010 A1 WO 9739010A1
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Prior art keywords
variant nucleotide
hpv
nucleic acid
nucleotide
variant
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PCT/US1997/006354
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French (fr)
Inventor
Cosette M. Wheeler
M. Michele Manos
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The University Of New Mexico
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Application filed by The University Of New Mexico filed Critical The University Of New Mexico
Priority to AU26725/97A priority Critical patent/AU2672597A/en
Priority to EP97918677A priority patent/EP1007538A4/en
Publication of WO1997039010A1 publication Critical patent/WO1997039010A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Definitions

  • the present invention relates probes constituted by labelled, mono-stranded DNA or RNA nucleic acid sequences.
  • HPVs Human papillomaviruses
  • HPV- 16 Human papillomaviruses
  • HPVs are characterized by a circular double strand DNA genome, about 8000 bases long, wrapped in a protein capsid 55-60 nm in diameter.
  • the virus can be found in non-malignant lesions in the unintegrated episomal state. In these cases disturbance of the cellular differentiation is observed and production of viral particles at late stages of the differentiation is observed.
  • Papillomaviruses are defined by genomic sequence similarities rather than by classical serology.
  • HPV genome is defined as a new type if it is separated by a Hamming distance or dissimilarity of more than 10% in its nucleotide sequence compared with other known HPV types in the E6, E7, and Ll open reading frames (ORFs) combined. Isolates within the same type differing by 0 to 2% in their nucleotide sequences compared with the reference sequence are referred to as variants, and those differing by 2 to 10% are referred to as subtypes.
  • Probes for HPVs made from DNA sequences may be obtained by various routes, particularly by genetic engineering or by manual or automatic direct synthesis. These nucleic acid sequences have the property of being matched and of forming hybrids with complementary DNA or RNA sequences, as the case may be denatured previously, if the latter were initially double stranded or mRNA. This denaturation can be done after incubation in a medium of high ionic strength and at high temperature or in a basic medium. These hybrids are then detectable.
  • the detection of hybrids can be done by different methods.
  • the probe may be labelled by one of the known methods for the labelling of nucleic acid probes. It may be radioactive labelling, for example, with phosphorus 32, or in the case of a cold probe, no radioactive labelling, for example enzymatic, as is also known. In certain cases, the probe is not labelled during its use proper, but modified chemically to be detectable after hybridization, for example with biotin.
  • HPV in the cervix is carried out by the use of molecular probes constituted by synthetic HPV-specific oligonucleotides or cloned viral DNA fragments that are labelled.
  • molecular probes constituted by synthetic HPV-specific oligonucleotides or cloned viral DNA fragments that are labelled.
  • synthetic DNA probes labelled non-isotopically or isotopically and usable in situ or in liquid or solid-phase assays.
  • there are identified at the level of the DNA specific regions of different types of HPV virus to deduce therefrom complementary homologous synthetic probes.
  • Figure 1 shows the genetic variation within HPV-18 in tabular form
  • Figure 2 shows the genetic variation within HPV-33 in tabular form
  • Figure 3 shows the genetic variation within HPV-35 in tabular form
  • Figure 4 shows the genetic variation within HPV-39 in tabular form
  • Figure 5 shows the genetic variation within HPV-45 in tabular form
  • Figure 6 shows the genetic variation within HPV-51 in tabular form
  • Figure 7 shows the genetic variation within HPV-52 in tabular form
  • Figure 8 shows the genetic variation within HPV-58 in tabular form
  • Figure 9 shows the genetic variation within HPV-59 in tabular form
  • Figure 10 shows the genetic variation within HPV-68 in tabular form
  • Figure 1 1 shows the genetic variation within MM4 in tabular form
  • Figure 12 shows the genetic variation within MM9/W13B in tabular form
  • Figure 13 is a table comparing intratypic nucleotide and amino acid sequence variation in the MY09/11 Ll region for 13 HPV types and novel sequences, including a reference type, HPV- 16.
  • RNA equivalent refers to a nucleic acid sequence substituting "U” (Uracil) for "T” in a corresponding DNA sequence.
  • Figures 1 to 12 present a nucleotide comparison with the corresponding reference HPV sequences for the 12 HPVs for which the present invention provides probes.
  • the first nucleotide of the specific HPV sequence has been used as nt 1 for each individual HPV type.
  • variations within a single HPV type or novel sequence range from no changes (HPV-33, HPV-35, HPV-45, HPV-51 HPV-52, HPV-58, HPV-59, HPV-68, and MM4) to a maximum of 19 changes (HPV-52).
  • Nucleotide changes between two individual specimens range from no changes (HPV-18, HPV-33, HPV-35, HPV-45, HPV-51, hPV- 52, HPV-58, HPV-59, HPV-68 and MM4) to a maximum of 9 changes in IS464 (an HPV-52 variant).
  • isolates can be classified into variant clusters.
  • Figure 13 is a table comparing intratypic nucleotide and amino acid sequence variation in the MY09/11 Ll region for the 12 HPV types of Figs. 1 to 12 and a reference type, HPV- 16
  • ORF Ll nucleotide positions at which variations have been observed are given across the top of each figure.
  • the numbering refers to the first nucleotide of each specific HPV genome for all HPV types except HPV-68, MM4, and MM9 for which the numbering refers to the first nucleotide of the sequence for each type as presented in the MY09/11 Ll fragment deposited in GenBank and as reported in a human papillomaviruses 1994 compendium, (Myers, G. et al.
  • HPV-73 The following HPVs were analyzed: HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51 , HPV-52, HPV- 58, HPV-59, HPV-68 (ME180), MM4/W13B (novel partial genomic sequence) and MM9/PAP238A (recently designated HPV-73).
  • PCR products were subjected to electrophoresis in 1 % Nusieve agarose 1 X TAE buffer and visualized under UV light Amplimers of the expected 450-bp size were excised from the gels and extracted with Gelase (Epicentre Technologies, Madison, WI) as described by the manufacturer. Gel- purified amplimers were ligated to a pGEM-T vector (Promega, Madison, WI). Individual transformants were subjected to colony direct PCR with Ml 3 forward and reverse p ⁇ mers.
  • Plasmid min ⁇ reps of cloned HPVs with correct insert size and positive by hyb ⁇ dization assays were prepared as desc ⁇ bed in Stewart, A., et al , 1995, Generation of Entire Human Papillomavirus Genomes by Long PCR and Frequency of Errors Produced in Amplification, Genome Research, 5:79-88.
  • Phylogenetic analyses were performed over the MY09/1 1 Ll region with 53 distinct HPV sequences.
  • the 53 HPV sequences were obtained from 53 separate clinical specimens. Twelve different HPV types and novel sequences and the co ⁇ esponding reference sequences were represented in the analyses For each type an alignment of the distinct MY09/11 sequences (Including the sequence of the reference clone from GenBank) was constructed by using the complete MY09/1 1 region except for the primer binding sites.
  • the most parsimonious phylogenetic trees were determined with PAUP 3.1.1 software on a Power Macintosh 7100 using the branch- and-bound search strategy, which is guaranteed to find the most parsimonious tree(s). In some cases, several equally parsimonious trees were obtained.
  • HPV-18 variants do not seem to form intermediate classes between the two major HPV-18 groups but may be representative of unique groups of HPV-18 variants.
  • HPV-18 reference does not account for variations at nt 6579, 6581, 6625, 6626, 6677, 6697, 6719, 6749, 6842, 6877, 6917, 6943, 6986 (nt 22, 24, 68, 69, 120, 140, 162, 192, 285, 320, 360, 386, 429 of SEQ ID NO: l below).
  • the present invention provides nucleic acid probes for HPV-18 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 21, 25 to 67, 70 to 1 19, 121 to 139, 163 to 191, 193 to 284, 286 to 319, 321 to 359, 361 to 385, 387 to 428 and 430 to 455 of SEQ ID NO: l and their RNA equivalents.
  • These probes avoid the region of variation in HPV-18 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-18 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:l which include a variant nucleotide located at at least one of numbered positions 22, 24, 68, 69, 120, 340, 162, 192, 285, 320, 360, 386, and 429 wherein: when the variant nucleotide is at nt 22, the variant nucleotide is A; when the variant nucleotide is at nt 24, the variant nucleotide is C; when the variant nucleotide is at nt 68, the variant nucleotide is G; when the variant nucleotide is at nt 69, the variant nucleotide is T or U; when the variant nucleotide is at nt 120, the variant nucleotide is G; when the variant nucleotide is at ntide is at
  • the present invention provides nucleic acid probes for HPV- 33 comprising an oligonucleotide of 18 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 98, 100 to 125, 127 to 165, 167 to 399 and 401 to 449 of SEQ ID NO:2 and their RNA equivalents.
  • These probes avoid the region of variation in HPV-33 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-33 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:2 which include a variant nucleotide located at at least one of numbered positions 99, 126, 166, and 400 wherein: when the variant nucleotide is at nt 99, the variant nucleotide is C; when the variant nucleotide is at nt 126, the variant nucleotide is G; when the variant nucleotide is at nt 166, the variant nucleotide is T; and when the variant nucleotide is at nt 400, the variant nucleotide is A; and RNA equivalents ofthe nucleic acid sequences.
  • These probes are capable of hybridizing with the HPV-33 variants shown in Figure 2.
  • the present invention provides nucleic acid probes for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 102 and 104 to 452 of SEQ ID NO:3 and their RNA equivalents. These probes avoid the region of variation in HPV-35 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:3 which include nt 103. These probes are capable of hybridizing with the HPV-35 variants shown in Figure 3.
  • Four of the specimens were prototype-like and had either one change each at nt 6638 (T to A) and 6903 (C to T) (ISO73 and IS270, respectively) or both of these changes (ISI 14 and IS281) (Fig. 4)
  • the remaining six specimens had three to six changes each .all but one (IS270) HPV-39 specimens had the C-to-T substitution at nt 6903, and none of the specimens were identical to the HPV-39 reference sequence.
  • HPV-39 reference does not account for variations at nt 6638, 6733, 6785, 6853, 6854, 6903, 6996 (54, 149, 201 , 269, 270, 319, and 412 of SEQ ID NO:4 below).
  • the present invention provides nucleic acid probes for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 53, 55 to 148, 150 to 200, 202 to 268, 271 to 318, 320 to 41 1 and 413 to 455 of SEQ ID NO:4 and their RNA equivalents.
  • These probes avoid the region of variation in HPV-33 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-39 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:4 which include a variant nucleotide located at at least one of numbered positions 54, 149, 201, 269, 270, 319 and 412 wherein: when the variant nucleotide is at nt 54, the variant nucleotide is A; when the variant nucleotide is at nt 149, the variant nucleotide is T; when the variant nucleotide is at nt 201 , the variant nucleotide is A; when the variant nucleotide is at nt 269, the variant nucleotide is C; when the variant nucleotide is at nt 270, the variant nucleotide is C; when the variant nucleotide is at nt 319, the variant
  • the remaining five specimens appear as an intermediate groups between the prototype/prototype-like group and the group containing African specimens sharing some of the maker substitutions from both groups. It was not possible to make any geographical assignments to any of the variants except for those of African origin, since the two other groups included specimens from various continents.
  • HPV-45 reference does not account for variations at nt 6621, 6661, 6665, 6676, 6677, 6687, 6705, 6816, 6837, 6852, 6861, 6862, 6910, 6914, 6951, 6996 (60, 100, 104, 1 15, 116, 126, 144, 255, 276, 291, 300, 301, 349, 353, 390, and 435 of SEQ ID NO:5 below).
  • the present invention provides nucleic acid probes for HPV-45 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 59, 61 to 99, 145 to 254, 256 to 275, 302 to 348, 354 to 389, 391 to 434 and 436 to 455 of SEQ ID NO:5 and their RNA equivalents.
  • These probes avoid the region of variation in HPV-45 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-45 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:5 which include a variant nucleotide located at at least one of numbered positions 60, 100, 104, 1 15, 116, 126, 144, 255, 276, 291, 300, 301, 349, 353, 390, and 435 wherein: when the variant nucleotide is at nt 60, the variant nucleotide is T; when the variant nucleotide is at nt 100, the variant nucleotide is G; when the variant nucleotide is at nt 104, the variant nucleotide is C; when the variant nucleotide is at nt 1 15, the variant nucleotide is G; when the variant nucleotide is at nt 116, the variant nucleotide is A
  • the present invention provides nucleic acid probes for HPV-51 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:6 which include nt 348. These probes are capable of hybridizing with the HPV-51 variants shown in Figure 6.
  • HPV-52 reference does not account for the variations at nt 6698, 6701, 6703, 6711, 6712, 6764, 6794, 6824, 6833, 6848, 6917, 6920, 6935, 6941, 6944, 6959, 6980, 6983, 6992 (96, 99, 101, 109, 110, 162, 192, 222, 231, 246, 315, 318, 333, 339, 342, 357, 378, 381, 390 of SEQ ID NO:7 below).
  • the present invention provides nucleic acid probes for HPV-52 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 95, 1 1 1 to 161 , 163 to 191 , 193 to 221, 247 to 314, 358 to 377 and 391 to 449 of SEQ ID NO:7 and their RNA equivalents.
  • These probes avoid the region of variation in HPV-52 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-52 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:7 which include a variant nucleotide located at at least one of numbered positions 96, 99, 101, 109; 1 10, 162, 192, 222, 231 , 246, 315, 318, 333, 339, 342, 357, 378, 381 , and 390 wherein: when the variant nucleotide is at nt 96, the variant nucleotide is A; when the variant nucleotide is at nt 99, the variant nucleotide is G; when the variant nucleotide is at nt 101, the variant nucleotide is C; when the variant nucleotide is at nt 109, the variant nucleotide is G; when the variant nucleotide is at n
  • Specimens within the HPV-58 group did not form any natural groups, with the exception of prototype or prototype-like (Fig.8). The majority of the samples (56%)) could be considered prototype or prototype-like, whereas the remaining sequences (44%) had three to eight nucleotide changes and share only one nucleotide substitution at position 240 (C to A). Overall, it has been found that the HPV-58 reference does not account for the variations at nt 6641, 6692, 6697, 671 1, 6798, 6822, 6827, 6828, 6881 , 7016 (54, 105, 110, 124, 211, 235, 240, 241 , 294, 429 of SEQ ID NO.8 below).
  • the present invention provides nucleic acid probes for HPV-58 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 53, 55 to 104, 125 to 210, 212 to 234, 242 to 293, 295 to 428 and 430 to 449 of SEQ ID NO:8 and their RNA equivalents.
  • These probes avoid the region of variation in HPV-58 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-58 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:8 which include a variant nucleotide located at at least one of numbered positions 54, 105, 110, 124, 21 1, 235, 240, 241, 294, and 429 wherein: when the variant nucleotide is at nt 54, the variant nucleotide is A; when the variant nucleotide is at nt 105, the variant nucleotide is A; when the variant nucleotide is at nt 110, the variant nucleotide is A; when the variant nucleotide is at nt 124, the variant nucleotide is A; when the variant nucleotide is at nt 21 1, the variant nucleotide is G; when the variant nucleotide is at nt 235
  • the present invention provides nucleic acid probes for HPV-59 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 65, 67 to 101, 104 to 399 and 401 to 455 of SEQ ID NO:9 and their RNA equivalents. These probes avoid the region of variation in HPV-59 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-59 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:9 which include a variant nucleotide located at at least one of numbered positions 66, 102, 103, and 400 wherein: when the variant nucleotide is at nt 66, the variant nucleotide is C; when the variant nucleotide is at nt 102, the variant nucleotide is G; when the variant nucleotide is at nt 103, the variant nucleotide is C or G; and when the variant nucleotide is at nt 400, the variant nucleotide is A; and RNA equivalents of the nucleic acid sequences.
  • These probes are capable of hybridizing with the HPV-59 variants shown in Figure 9.
  • the present invention provides nucleic acid probes for HPV-68 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 209, 1 1 1 to 344, 346 to 377 and 379 to 455 of SEQ ID NO: 10 and their RNA equivalents.
  • These probes avoid the region of variation in HPV-68 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for HPV-68 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO: 10 which include a variant nucleotide located at at least one of numbered positions 210, 345 and 378 wherein: when the variant nucleotide is at nt 210, the variant nucleotide is A; when the variant nucleotide is at nt 345, the variant nucleotide is G; and when the variant nucleotide is at nt 378, the variant nucleotide is C; and RNA equivalents of the nucleic acid sequences.
  • These probes are capable of hybridizing with the HPV-68 variants shown in Figure 10.
  • the present invention provides nucleic acid probes for MM4 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 11 1 , 1 13 to 139, 145 to 248, 250 to 267, 269 to 290, 292 to 369 and 317 to 455 of SEQ ID NO:l 1 and their RNA equivalents.
  • These probes avoid the region of variation in MM4 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for MM4 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:l l which include a variant nucleotide located at at least one of numbered positions 1 12, 140, 144, 249, 268, 291 , and 370 wherein: when the variant nucleotide is at nt 1 12, the variant nucleotide is A; when the variant nucleotide is at nt 140, the variant nucleotide is G; when the variant nucleotide is at nt 144, the variant nucleotide is G or T; when the variant nucleotide is at nt 249, the variant nucleotide is C; when the variant nucleotide is at nt 268, the variant nucleotide is C; when the variant nucleotide is at nt 291, the variant nucleotide
  • the present invention provides nucleic acid probes for MM9 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences from numbered positions 1 to 101, 102 to 143, 145 to 169, 188 to 302, 304 to 334, 336 to 374, 376 to 416 and 418 to 458 of SEQ ID NO: 12 and their RNA equivalents.
  • These probes avoid the region of variation in MM9 while providing a probe of a convenient size for conventional hybridization techniques.
  • the present invention provides nucleic acid probes for MM9 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO: 12 which include a variant nucleotide located at at least one of numbered positions 102, 144, 170, 187, 303, 333, 375, and 417 wherein: when the variant nucleotide is at nt 102, the variant nucleotide is A; when the variant nucleotide is at nt 144, the variant nucleotide is A; when the variant nucleotide is at nt 170, the variant nucleotide is A; when the variant nucleotide is at nt 187, the variant nucleotide is T; when the variant nucleotide is at nt 303, the variant nucleotide is G; when the variant nucleotide is at ntide
  • a desired sequence region of all known HPV types is aligned using a conventional nucleic acid alignment program, such as the PileUp program produced by the Wisconsin Genetics Computer Group (GCG).
  • GCG Wisconsin Genetics Computer Group
  • Potential type-specific oligonucleotide probes are marked using the following criteria: length of 15 to 30 bp, preferably 18 to 22 bp; preferably a G/C to A/T ratio of 50:50, if possible; preferably no strings of consecutive A, T or A and T longer than 4, if possible;
  • One G or C is weighted as 4°C
  • One A or T is weighted as 2°C
  • probes are compared against all known HPV sequences in the selected regions.
  • a suitable program is the FASTA program of GCG.
  • probes having at least four nucleotide mismatches when compared with all known HPV types are chosen, with a preference for probes with greater than four mismatches.
  • probes produced by this process are designed for a predicted hybridization temperature (50 to 55°C), this procedure can be changed slightly for other hybridization parameters. For example, a lower hybridization temperature will require the oligonucleotide type- or allele-specific probe to have a greater number of mismatches and different length.
  • A represents adenine
  • C represents cytosine
  • G represents guanine
  • T represents thymine
  • U represents uracil
  • M represents A or C
  • R represents A or G
  • W represents A or T/U
  • S represents C or G
  • Y represents C or T/U
  • K represents G or T/U
  • V represents A or C or G, not T/U
  • H represents A or C or T/U, not G
  • D represents A or G or T/U, not C
  • B represents C or G or T/U, not A
  • N represents (A or C or G or T/U) or (unknown or other).
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAACTATGCA AAGTTACCTT AACTGCAGAA GTTATGACAT ATATTCATGC TATGAATCCA 240
  • MOLECULE TYPE DNA (genomic)
  • GCACAAGGCC ATAATAATGG TATTTGTTGG AGTAACCAAT TGTTTGTTAC TGTAGTTGAT 60
  • MOLECULE TYPE DNA (genomic)
  • AATTCCTCTA TATTGGACAA TTGGAATTYY GCTGTAGCTC CTCCACCATC TGCCAGTTTG 300
  • MOLECULE TYPE DNA (genomic)
  • GATTCTACMA TTTTAGAACA GTGGAATYTT GGATTAACCT TGCCCCCCTC MGCTAGTTTG 300
  • CAGGCTAAAS AAGACCCTTT GGCAAAATAT AAATTTTGGA ATGTAGACCT TAAGGAACGC 420
  • MOLECULE TYPE DNA (genomic)

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Abstract

The present invention provides probes for the human papillomaviruses HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM4/W13B and MM9/PAP238A which hybridize with the homologous regions of these HPVs' DNA. The present invention also provides probes for these human papillomaviruses which are capable of hybridizing with newly discovered variant regions of these HPVs' DNA.

Description

PROBES FOR THE DETECTION OF HUMAN PAPILLOMAVIRUS
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates probes constituted by labelled, mono-stranded DNA or RNA nucleic acid sequences.
Description of the Prior Art
Human papillomaviruses (HPVs) constitute a group of viruses associated with benign and malignant neoplasia of cutaneous and mucosal epithelia. To date, more than 70 different HPV types have been identified. Evidence from partial sequences suggest the existence of at least 13 additional types that would qualify as novel HPVs, and it is likely that unidentified HPVs remain. About half of the reported HPVs are associated with mucosal lesions, including cervical neoplasia. A strong and consistent association has been bound between infection with certain types of HPVs (e.g., HPV- 16) and invasive cervical carcinoma. HPV- 16 represents about 50% of the cervical cancer- associated HPV infections worldwide, although regional variations have been reported.
HPVs are characterized by a circular double strand DNA genome, about 8000 bases long, wrapped in a protein capsid 55-60 nm in diameter. The virus can be found in non-malignant lesions in the unintegrated episomal state. In these cases disturbance of the cellular differentiation is observed and production of viral particles at late stages of the differentiation is observed. When uterine cervical carcinoma is established, a variable integration of certain sequences of viral DNA in the cellular DNA is often but not always observed. Papillomaviruses (PVs) are defined by genomic sequence similarities rather than by classical serology. An HPV genome is defined as a new type if it is separated by a Hamming distance or dissimilarity of more than 10% in its nucleotide sequence compared with other known HPV types in the E6, E7, and Ll open reading frames (ORFs) combined. Isolates within the same type differing by 0 to 2% in their nucleotide sequences compared with the reference sequence are referred to as variants, and those differing by 2 to 10% are referred to as subtypes.
Probes for HPVs made from DNA sequences may be obtained by various routes, particularly by genetic engineering or by manual or automatic direct synthesis. These nucleic acid sequences have the property of being matched and of forming hybrids with complementary DNA or RNA sequences, as the case may be denatured previously, if the latter were initially double stranded or mRNA. This denaturation can be done after incubation in a medium of high ionic strength and at high temperature or in a basic medium. These hybrids are then detectable.
The detection of hybrids can be done by different methods. The probe may be labelled by one of the known methods for the labelling of nucleic acid probes. It may be radioactive labelling, for example, with phosphorus 32, or in the case of a cold probe, no radioactive labelling, for example enzymatic, as is also known. In certain cases, the probe is not labelled during its use proper, but modified chemically to be detectable after hybridization, for example with biotin.
The detection of HPV in the cervix is carried out by the use of molecular probes constituted by synthetic HPV-specific oligonucleotides or cloned viral DNA fragments that are labelled. Within the framework of epidemiological studies and routine diagnosis of infections by HPV, it is useful to develop a rapid, simple, specific and sensitive method based on the use of synthetic DNA probes labelled non-isotopically or isotopically and usable in situ or in liquid or solid-phase assays. To this end there are identified at the level of the DNA, specific regions of different types of HPV virus to deduce therefrom complementary homologous synthetic probes. SUMMARY OF THE INVENTION
It has been discovered that currently available probes for a number of types of HPVs are deficient, because they are designed to hybridize with sections of the HPV where there are genetic variations within the subtypes and variants of a given HPV type. For this reason, these probes may not be completely effective in hybridizing with all of the HPVs of a given type in a sample being tested.
Therefore, in one preferred embodiment, it is an object of the present invention to provide probes for the human papillomaviruses HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM4/W13B (novel partial genomic sequence) and MM9/PAP238A (designated HPV-73) which hybridize with the homologous regions of these HPVs' DNA.
In a second preferred embodiment, it is an object of the present invention to provide probes for variants of the human papillomaviruses HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM4/W13B (novel partial genomic sequence) and MM9/PAP238 (designated HPV-73) which hybridize with newly discovered variant regions of these HPVs' DNA.
Other objects and features ofthe present invention will be apparent from the following detailed description of the preferred embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described in conjunction with the accompanying drawings, in which: Figure 1 shows the genetic variation within HPV-18 in tabular form;
Figure 2 shows the genetic variation within HPV-33 in tabular form;
Figure 3 shows the genetic variation within HPV-35 in tabular form;
Figure 4 shows the genetic variation within HPV-39 in tabular form;
Figure 5 shows the genetic variation within HPV-45 in tabular form;
Figure 6 shows the genetic variation within HPV-51 in tabular form;
Figure 7 shows the genetic variation within HPV-52 in tabular form;
Figure 8 shows the genetic variation within HPV-58 in tabular form;
Figure 9 shows the genetic variation within HPV-59 in tabular form;
Figure 10 shows the genetic variation within HPV-68 in tabular form;
Figure 1 1 shows the genetic variation within MM4 in tabular form;
Figure 12 shows the genetic variation within MM9/W13B in tabular form; and
Figure 13 is a table comparing intratypic nucleotide and amino acid sequence variation in the MY09/11 Ll region for 13 HPV types and novel sequences, including a reference type, HPV- 16.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
For the purposes of the present invention, the term "RNA equivalent" refers to a nucleic acid sequence substituting "U" (Uracil) for "T" in a corresponding DNA sequence.
Figures 1 to 12 present a nucleotide comparison with the corresponding reference HPV sequences for the 12 HPVs for which the present invention provides probes. For the purposes of orientation, the first nucleotide of the specific HPV sequence has been used as nt 1 for each individual HPV type. When considering all nucleotide substitutions in all MY09/11 variants (excluding 5' and 3' primer regions), variations within a single HPV type or novel sequence range from no changes (HPV-33, HPV-35, HPV-45, HPV-51 HPV-52, HPV-58, HPV-59, HPV-68, and MM4) to a maximum of 19 changes (HPV-52). Nucleotide changes between two individual specimens range from no changes (HPV-18, HPV-33, HPV-35, HPV-45, HPV-51, hPV- 52, HPV-58, HPV-59, HPV-68 and MM4) to a maximum of 9 changes in IS464 (an HPV-52 variant). In some of these HPV types analyzed, isolates can be classified into variant clusters.
Figure 13 is a table comparing intratypic nucleotide and amino acid sequence variation in the MY09/11 Ll region for the 12 HPV types of Figs. 1 to 12 and a reference type, HPV- 16
In Figures 1 to 12, ORF Ll nucleotide positions at which variations have been observed are given across the top of each figure. The numbering refers to the first nucleotide of each specific HPV genome for all HPV types except HPV-68, MM4, and MM9 for which the numbering refers to the first nucleotide of the sequence for each type as presented in the MY09/11 Ll fragment deposited in GenBank and as reported in a human papillomaviruses 1994 compendium, (Myers, G. et al. (ed.), 1994, Human Papillomaviruses, A Compilation and Analysis of Nucleic Acid Sequences, Publication LA-UR 94-0000, Los Alamos National Laboratory, Los Alamos, N. Mexico). The reference sequence used for each type is indicated as "ref. Each row in each panel of each figure indicates, from left to right, the following information: the specimen identification codes, the nucleotide sequence alignment compared with the reference sequence, and the origin of each specimen on the right. Specimens obtained from the IBSCC, (Bosch, F.X. et al, 1995, Prevalence of Human Papillomavirus in Cervical Cancer: A Worldwide Perspective, J. National Cancer Inst., 87:796-802), begin with the letters IS. All remaining specimens were obtained from studies performed in Albuquerque, N.M. For each variant sequence, positions that do not vary relative to the HPV reference nucleotide or amino acid sequence are marked with a dash (-) in the alignment. Nucleotide changes resulting in nonsynonymous amino acid changes are indicated above the reference sequence for each HPV type with an asterisk (*). The symbol (*) in Figure 1 indicates a corrected nonsynonymous amino acid change as a result of a sequencing error detected in the HPV-18 reference sequence.
The specimens in Figures 1 to 12 include invasive cervical tumor biopsy specimens («=135) (Bosch, F.X., et al, 1995, Prevalence of Human Papillomavirus in Cervical Cancer: A Worldwide Perspective, J. National Cancer Inst., 87:796-802) or cervical swab specimens («=1 1) (Becker, T.M., et al, 1994, Sexually Transmitted Diseases and Other Risk Factors for Cervical Dysplasia Among Southwestern Hispanic and Non-Hispanic White Women, JAMA, 271 : 1 181-1 188; and Wheeler, CM., et al, 1993, Determinants of Genital Human Papillomavirus Infection Among Cytogically Normal Women Attending the University of New Mexico Student Health Center, Sexually Transmitted Dis. 20:286-289). Crude proteinase digests were prepared and specific HPV types were determined for each epidemiological investigation as described in Becker, T.M., et al, 1994, Sexually Transmitted Diseases and Other Risk Factors for Cervical Dysplasia Among Southwestern Hispanic and Non-Hispanic White Women, JAMA, 271 : 1 181-1 188; Bosch, F.X., et al, 1995, Prevalence of Human Papillomavirus in Cervical Cancer: A Worldwide Perspective, J. National Cancer Inst., 87:796-802; and Wheeler, CM., et al, 1993, Determinants of Genital Human Papillomavirus Infection Among Cytogically Normal Women Attending the University of New Mexico Student Health Center, Sexually Transmitted Dis. 20:286-289). The following HPVs were analyzed: HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51 , HPV-52, HPV- 58, HPV-59, HPV-68 (ME180), MM4/W13B (novel partial genomic sequence) and MM9/PAP238A (recently designated HPV-73). The number of specimens analyzed for each HPV type and novel sequence is shown in Figure 13. Sequences for all reference HPVs are deposited in GenBank and are listed in the compendium, (Myers, G., et al. (ed.), 1994, Human Papillomaviruses, A Compilation and Analysis of Nucleic Acid Sequences, Publication LA-UR 94-0000, Los Alamos National Laboratory, Los Alamos, N. Mexico).
Amplification of a ~450-by HPV specific segment from the Ll ORF was performed as described in Bauer, H.M., et al, 1992, Determination of Genital HPV Infection Using Consensus PCR, p. 131 -152, ln CS. Herrington and J.O.D. McGee (cd.), Diagnostic Molecular Pathology: A Practical Approach, Oxford University Press, Oxford, UK, with the MY09/1 1 Ll consensus primer system described in Bauer, H.M. et al, 1992, Determination of Genital HPV Infection Using Consensus PCR, p. 131 - 152, In CS. Herrington and J.O.D. McGee (ed.) Diagnostic Molecular Pathology: A Practical Approach, Oxford University Press, Oxford, UK. This region corresponds in the HPV- 16 genome to nucleotides (nt) 6580 to 7030. PCR products were subjected to electrophoresis in 1 % Nusieve agarose 1 X TAE buffer and visualized under UV light Amplimers of the expected 450-bp size were excised from the gels and extracted with Gelase (Epicentre Technologies, Madison, WI) as described by the manufacturer. Gel- purified amplimers were ligated to a pGEM-T vector (Promega, Madison, WI). Individual transformants were subjected to colony direct PCR with Ml 3 forward and reverse pπmers. Resultant PCR products were subsequently screened by hybπdization with HPV type-specific probes descπbed in (Bauer, H.M. et al , 1992, Determination of Genital HPV Infection Using Consensus PCR, p. 131-152, In CS. Herπngton and J.O.D. McGee (ed.) Diagnostic Molecular Pathology A Practical Approach, Oxford University Press, Oxford, UK; and Hildesheim, A., et al, 1994, Persistence of Type- Specific Human Papillomavirus Infection Among Cytogically Normal Women, J Infect
Figure imgf000009_0001
Plasmid minφreps of cloned HPVs with correct insert size and positive by hybπdization assays were prepared as descπbed in Stewart, A., et al , 1995, Generation of Entire Human Papillomavirus Genomes by Long PCR and Frequency of Errors Produced in Amplification, Genome Research, 5:79-88. At least two clones for each clinical specimen were subsequently subjected to dideoxy sequencing as described in Sanger, F., et al , 1977, DNA Sequencing with Chain-Terminating Inhibitors, Proc National Acad Sci., USA, 74:5463-5467, with 35S-dATP and Sequenase 2.0 (U.S Biochemical, Cleveland, OH). Both forward- and reverse-strand sequences were obtained for each clone. Specific sequencing pπmers in the pGEM-T vector, pGEM-F (5'-ACGGCCAGTGAATTGTA-3') and pGEM-B (5'-ACGCGTTGGGAGCTCTC-3') were used to facilitate sequencing efforts. PGEM-F and pGEM-R are closer to the pGEM-T insertion site than are standard Ml 3 sequencing primers
Phylogenetic analyses were performed over the MY09/1 1 Ll region with 53 distinct HPV sequences. The 53 HPV sequences were obtained from 53 separate clinical specimens. Twelve different HPV types and novel sequences and the coπesponding reference sequences were represented in the analyses For each type an alignment of the distinct MY09/11 sequences (Including the sequence of the reference clone from GenBank) was constructed by using the complete MY09/1 1 region except for the primer binding sites. The most parsimonious phylogenetic trees were determined with PAUP 3.1.1 software on a Power Macintosh 7100 using the branch- and-bound search strategy, which is guaranteed to find the most parsimonious tree(s). In some cases, several equally parsimonious trees were obtained.
The nucleotide sequences described herein have been submitted to GenBank. Accession number have been assigned as follows: U45889 to U45894 to the HPV-18 sequences, U45895 to U45897 to the HPV-33 sequences, U45898 to the HPV-35 sequence, U45899 to U45905 to the HPV-39 sequences, U45906 to U45916 to the HPV-45 sequences, U45917 to the HPV-51 sequence, U45918 to U45923 to the HPV- 52 sequences, U5924 to U45929 to the HPV-58 sequences, U45930 to U45933 to the HPV-59 sequences, U45934 to the HPV-68(ME180) sequence, U45935 to U45937 to the MM9 sequences, and U45939 to the MM4 sequence, IS 1019. Additional MM4 sequences including IS766, IS887, and ISO 16 were previously assigned GenBank accession numbers U12483, U12484, and U12482, respectively.
In the 44 samples of HPV-18, two main groups were observed, one prototype or prototype-like and one with variants containing six or more changes compared with the reference sequence (Fig. 13). In the following text, the word "prototype" or "prototype¬ like" refers to the coπesponding reference clone and published sequence of the original HPV isolate. The majority (77%) of the HPV-18 specimens analyzed fell into the prototype group. One specimen from Chile, IS326, had two substitutions at nt positions 6677 and 6697, that were not observed in any of the other HPV 18 specimens analyzed, a T-to-C substitution at position 6877 and an A-to-G substitution at position 6943. These two HPV-18 variants do not seem to form intermediate classes between the two major HPV-18 groups but may be representative of unique groups of HPV-18 variants. The nine remaining HPV-18 specimens (21 %) all had six or more changes, and all but one (IS227 from Cuba) were obtained in Africa.
None of the HPV-18 specimens analyzed (Fig. 1) contained the previously reported HPV reference sequence. All 44 of the HPV-18 specimens analyzed had two nucleotide changes, a C-to-G substitution at position 6625, resulting in a substitution (proline to arginine) at amino acid (aa) 399 in the Ll ORF, and a synonymous C-to-G substitution at nt 6842. The nucleotide substitution at position 6625 has been previously reported, whereas the nucleotide change at position 6842 has not. These changes could reflect the extreme rarity of the reference sequence that originally was obtained from a subject in northeastern Brazil. Alternatively, these changes could be due to cloning artifacts or sequencing eπors in the original HPV-18 reference clone. Sequence analysis of the reference clone obtained from the DKFZ Papillomavirus Referenzzentrum, Heidelberg, Germany, revealed that this clone also had the two nucleotide changes at positions 6625 and 6842. Consequently, the previously reported HPV-18 reference has two sequencing eπors at nt 6625 and 6842.
Overall, it has been found that the HPV-18 reference does not account for variations at nt 6579, 6581, 6625, 6626, 6677, 6697, 6719, 6749, 6842, 6877, 6917, 6943, 6986 (nt 22, 24, 68, 69, 120, 140, 162, 192, 285, 320, 360, 386, 429 of SEQ ID NO: l below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-18 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 21, 25 to 67, 70 to 1 19, 121 to 139, 163 to 191, 193 to 284, 286 to 319, 321 to 359, 361 to 385, 387 to 428 and 430 to 455 of SEQ ID NO: l and their RNA equivalents. These probes avoid the region of variation in HPV-18 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-18 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:l which include a variant nucleotide located at at least one of numbered positions 22, 24, 68, 69, 120, 340, 162, 192, 285, 320, 360, 386, and 429 wherein: when the variant nucleotide is at nt 22, the variant nucleotide is A; when the variant nucleotide is at nt 24, the variant nucleotide is C; when the variant nucleotide is at nt 68, the variant nucleotide is G; when the variant nucleotide is at nt 69, the variant nucleotide is T or U; when the variant nucleotide is at nt 120, the variant nucleotide is G; when the variant nucleotide is at nt 140, the variant nucleotide is G; when the variant nucleotide is at nt 162, the variant nucleotide is A; when the variant nucleotide is at nt 192, the variant nucleotide is A; when the variant nucleotide is at nt 285, the variant nucleotide is G; when the variant nucleotide is at nt 320, the variant nucleotide is C; when the variant nucleotide is at nt 386, the variant nucleotide is C or G; and when the variant nucleotide is at nt 32, the variant nucleotide is G; and RNA equivalents ofthe nucleic acid sequences. These probes are capable of hybridizing with the HPV-18 variants shown in Figure 1.
HPV-33 specimens (n = 10) with four nucleotide substitutions observed in HPV- 33 (n =4) (Fig. 2). The remaining specimens are all identical to the reference sequence. Overall, it has been found that the HPV-33 reference does not account for variations at nt 6637, 6664, 6704, 6938 (99, 126, 166, 400 in SEQ ID NO:2 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV- 33 comprising an oligonucleotide of 18 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 98, 100 to 125, 127 to 165, 167 to 399 and 401 to 449 of SEQ ID NO:2 and their RNA equivalents. These probes avoid the region of variation in HPV-33 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment the present invention provides nucleic acid probes for HPV-33 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:2 which include a variant nucleotide located at at least one of numbered positions 99, 126, 166, and 400 wherein: when the variant nucleotide is at nt 99, the variant nucleotide is C; when the variant nucleotide is at nt 126, the variant nucleotide is G; when the variant nucleotide is at nt 166, the variant nucleotide is T; and when the variant nucleotide is at nt 400, the variant nucleotide is A; and RNA equivalents ofthe nucleic acid sequences. These probes are capable of hybridizing with the HPV-33 variants shown in Figure 2.
HPV-35 (/. = 8) shows limited variation, with a single nucleotide substitution identified in HPV-35 (n = 6) (Fig. 3) The remaining are all identical to the reference sequence. Overall, it has been found that the HPV-35 reference does not account for variation at nt 6624 (103 in SEQ ID NO:3 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 102 and 104 to 452 of SEQ ID NO:3 and their RNA equivalents. These probes avoid the region of variation in HPV-35 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:3 which include nt 103. These probes are capable of hybridizing with the HPV-35 variants shown in Figure 3.
The HPV-39 specimens (n = 10) analyzed did not form any obvious discrete groups. Four of the specimens were prototype-like and had either one change each at nt 6638 (T to A) and 6903 (C to T) (ISO73 and IS270, respectively) or both of these changes (ISI 14 and IS281) (Fig. 4) The remaining six specimens had three to six changes each .all but one (IS270) HPV-39 specimens had the C-to-T substitution at nt 6903, and none of the specimens were identical to the HPV-39 reference sequence. Overall, it has been found that the HPV-39 reference does not account for variations at nt 6638, 6733, 6785, 6853, 6854, 6903, 6996 (54, 149, 201 , 269, 270, 319, and 412 of SEQ ID NO:4 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 53, 55 to 148, 150 to 200, 202 to 268, 271 to 318, 320 to 41 1 and 413 to 455 of SEQ ID NO:4 and their RNA equivalents. These probes avoid the region of variation in HPV-33 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-39 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:4 which include a variant nucleotide located at at least one of numbered positions 54, 149, 201, 269, 270, 319 and 412 wherein: when the variant nucleotide is at nt 54, the variant nucleotide is A; when the variant nucleotide is at nt 149, the variant nucleotide is T; when the variant nucleotide is at nt 201 , the variant nucleotide is A; when the variant nucleotide is at nt 269, the variant nucleotide is C; when the variant nucleotide is at nt 270, the variant nucleotide is C; when the variant nucleotide is at nt 319, the variant nucleotide is T or U; and when the variant nucleotide is at nt 412, the variant nucleotide is C; and RNA equivalents ofthe nucleic acid sequences. These probes are capable of hybridizing with the HPV-39 variants shown in Figure 4.
HPV-45 specimens (n = 21) could be classified into at least three major groups (Fig. 5). Five of the specimens (23%) were classified as prototype or prototype-like. All variants within this group, except one that had a G-to-A change at nt 6861 , had no substitution when compared with the HPV-45 reference sequence. The largest (36%) group of HPV-45 variants had six to eight substitutions each, with six of these changes (at nt 6677, 6687, 6705, 6816, 6837, and 6861) being common to all of these variants. Four HPV-45 variants (18%) that were observed solely in African specimens shared two "marker" substitutions at nt 6665 and 6914. The remaining five specimens appear as an intermediate groups between the prototype/prototype-like group and the group containing African specimens sharing some of the maker substitutions from both groups. It was not possible to make any geographical assignments to any of the variants except for those of African origin, since the two other groups included specimens from various continents.
Overall, it has been found that the HPV-45 reference does not account for variations at nt 6621, 6661, 6665, 6676, 6677, 6687, 6705, 6816, 6837, 6852, 6861, 6862, 6910, 6914, 6951, 6996 (60, 100, 104, 1 15, 116, 126, 144, 255, 276, 291, 300, 301, 349, 353, 390, and 435 of SEQ ID NO:5 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-45 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 59, 61 to 99, 145 to 254, 256 to 275, 302 to 348, 354 to 389, 391 to 434 and 436 to 455 of SEQ ID NO:5 and their RNA equivalents. These probes avoid the region of variation in HPV-45 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-45 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:5 which include a variant nucleotide located at at least one of numbered positions 60, 100, 104, 1 15, 116, 126, 144, 255, 276, 291, 300, 301, 349, 353, 390, and 435 wherein: when the variant nucleotide is at nt 60, the variant nucleotide is T; when the variant nucleotide is at nt 100, the variant nucleotide is G; when the variant nucleotide is at nt 104, the variant nucleotide is C; when the variant nucleotide is at nt 1 15, the variant nucleotide is G; when the variant nucleotide is at nt 116, the variant nucleotide is A; when the variant nucleotide is at nt 126, the variant nucleotide is T or U; when the variant nucleotide is at nt 144, the variant nucleotide is A or C; when the variant nucleotide is at nt 255, the variant nucleotide is G; when the variant nucleotide is at nt 276, the variant nucleotide is A or G; when the variant nucleotide is at nt 291, the variant nucleotide is C; when the variant nucleotide is at nt 300, the variant nucleotide is A; when the variant nucleotide is at nt 301, the variant nucleotide is A; when the variant nucleotide is at nt 349, the variant nucleotide is A; when the variant nucleotide is at nt 353, the variant nucleotide is G; when the variant nucleotide is at nt 390, the variant nucleotide is G; and when the variant nucleotide is at nt 435, the variant nucleotide is A; and RNA equivalents of the nucleic acid sequences. These probes are capable of hybridizing with the HPV-45 variants shown in Figure 5.
HPV-51 (n - 6) shows limited variation, with a single nucleotide substitution HPV-51 (n = 1 ; t273), respectively (Fig. 6). The remaining are all identical to the reference sequence. Overall, it has been found that the HPV-51 reference does not account for the variation at nt 6813 (348 in SEQ ID NO:6 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-51 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 347 and 349 to 452 of SEQ ID NO:6 and their RNA equivalents. These probes avoid the region of variation in HPV-51 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-51 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:6 which include nt 348. These probes are capable of hybridizing with the HPV-51 variants shown in Figure 6.
HPV-52 specimens (n =13) showed a great number of intratype nucleotide substitutions (Fig. 7). A total of 19 substitution were observed in all HPV-52 variants combined. Of the HPV-52 specimens, 60% could be classified as prototype or prototype-like; all these specimens had no nucleotide changes (n = 7) or two changes (n = 1) (IS05). The remaining five specimens had between 4 and 10 changes each. Only one substitution, C to A at nt 6917, was shared among these six HPV-52 sequences. Overall, it has been found that the HPV-52 reference does not account for the variations at nt 6698, 6701, 6703, 6711, 6712, 6764, 6794, 6824, 6833, 6848, 6917, 6920, 6935, 6941, 6944, 6959, 6980, 6983, 6992 (96, 99, 101, 109, 110, 162, 192, 222, 231, 246, 315, 318, 333, 339, 342, 357, 378, 381, 390 of SEQ ID NO:7 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-52 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 95, 1 1 1 to 161 , 163 to 191 , 193 to 221, 247 to 314, 358 to 377 and 391 to 449 of SEQ ID NO:7 and their RNA equivalents. These probes avoid the region of variation in HPV-52 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-52 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:7 which include a variant nucleotide located at at least one of numbered positions 96, 99, 101, 109; 1 10, 162, 192, 222, 231 , 246, 315, 318, 333, 339, 342, 357, 378, 381 , and 390 wherein: when the variant nucleotide is at nt 96, the variant nucleotide is A; when the variant nucleotide is at nt 99, the variant nucleotide is G; when the variant nucleotide is at nt 101, the variant nucleotide is C; when the variant nucleotide is at nt 109, the variant nucleotide is G; when the variant nucleotide is at nt 110, the variant nucleotide is A; when the variant nucleotide is at nt 162, the variant nucleotide is C; when the variant nucleotide is at nt 192, the variant nucleotide is G; when the variant nucleotide is at nt 222, the variant nucleotide is T; when the variant nucleotide is at nt 231, the variant nucleotide is A; when the variant nucleotide is at nt 246, the variant nucleotide is C; when the variant nucleotide is at nt 315, the variant nucleotide is A; when the variant nucleotide is at nt 318, the variant nucleotide is G; when the variant nucleotide is at nt 333, the variant nucleotide is A; when the variant nucleotide is at nt 339, the variant nucleotide is G; when the variant nucleotide is at nt 342, the variant nucleotide is G; when the variant nucleotide is at nt 357, the variant nucleotide is G; when the variant nucleotide is at nt 378, the variant nucleotide is A; when the variant nucleotide is at nt 381, the variant nucleotide is G; and when the variant nucleotide is at nt 390, the variant nucleotide is C; and RNA equivalents of the nucleic acid sequences. These probes are capable of hybridizing with the HPV-52 variants shown in Figure 7.
Specimens within the HPV-58 group (n = 9) did not form any natural groups, with the exception of prototype or prototype-like (Fig.8). The majority of the samples (56%)) could be considered prototype or prototype-like, whereas the remaining sequences (44%) had three to eight nucleotide changes and share only one nucleotide substitution at position 240 (C to A). Overall, it has been found that the HPV-58 reference does not account for the variations at nt 6641, 6692, 6697, 671 1, 6798, 6822, 6827, 6828, 6881 , 7016 (54, 105, 110, 124, 211, 235, 240, 241 , 294, 429 of SEQ ID NO.8 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-58 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 53, 55 to 104, 125 to 210, 212 to 234, 242 to 293, 295 to 428 and 430 to 449 of SEQ ID NO:8 and their RNA equivalents. These probes avoid the region of variation in HPV-58 while providing a probe of a convenient size for conventional hybridization techniques. In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-58 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:8 which include a variant nucleotide located at at least one of numbered positions 54, 105, 110, 124, 21 1, 235, 240, 241, 294, and 429 wherein: when the variant nucleotide is at nt 54, the variant nucleotide is A; when the variant nucleotide is at nt 105, the variant nucleotide is A; when the variant nucleotide is at nt 110, the variant nucleotide is A; when the variant nucleotide is at nt 124, the variant nucleotide is A; when the variant nucleotide is at nt 21 1, the variant nucleotide is G; when the variant nucleotide is at nt 235, the variant nucleotide is A; when the variant nucleotide is at nt 240, the variant nucleotide is A; when the variant nucleotide is at nt 241, the variant nucleotide is G; when the variant nucleotide is at nt 294, the variant nucleotide is G; and when the variant nucleotide is at nt 429, the variant nucleotide is G; and RNA equivalents of the nucleic acid sequences. These probes are capable of hybridizing with the HPV-58 variants shown in Figure 8.
HPV-59 specimens (n = 11) showed very limited polymorphism (Fig. 9). Two specimens (18%) had no substitutions compared with the reference sequence. Four HPV-59 specimens had two substitutions at nt 6653 (T to C) and 6950 (C to A) and formed the largest group of HPV-59 variants. Another four HPV-59 specimens shared the C-to-A substitution with the former HPV-59 group but had a G rather than the C substitution at nt 6653. One of these specimens, IS583, had an additional T-to-C change at nt 6616 that was not detected in any of the other HPV-59 specimens analyzed. One specimen IS598, also shared the C-to-A nucleotide change observed in the two former HPV-59 groups but had an additional nucleotide change at position 6652 (T to G) that was not observed in any of the other HPV-59 specimens analyzed.
Overall, it has been found that the HPV-59 reference does not account for the variations at nt 6616, 6652, 6653, 6950 (66, 102, 103, 400 of SEQ ID NO:9 below). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-59 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 65, 67 to 101, 104 to 399 and 401 to 455 of SEQ ID NO:9 and their RNA equivalents. These probes avoid the region of variation in HPV-59 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-59 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:9 which include a variant nucleotide located at at least one of numbered positions 66, 102, 103, and 400 wherein: when the variant nucleotide is at nt 66, the variant nucleotide is C; when the variant nucleotide is at nt 102, the variant nucleotide is G; when the variant nucleotide is at nt 103, the variant nucleotide is C or G; and when the variant nucleotide is at nt 400, the variant nucleotide is A; and RNA equivalents of the nucleic acid sequences. These probes are capable of hybridizing with the HPV-59 variants shown in Figure 9.
HPV-68 (ME-180) (n = 6) shows limited total nucleotide variation with three nucleotides substitutions observed in HPV-68 (n = 1; IS362) (Fig. 10). The remaining specimens are all identical to the reference sequence. Overall, it has been found that the HPV-68 reference does not account for the variations at 2771, 2906, 2939 (210, 345, 378 of SEQ ID NO: 10). Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for HPV-68 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 209, 1 1 1 to 344, 346 to 377 and 379 to 455 of SEQ ID NO: 10 and their RNA equivalents. These probes avoid the region of variation in HPV-68 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for HPV-68 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO: 10 which include a variant nucleotide located at at least one of numbered positions 210, 345 and 378 wherein: when the variant nucleotide is at nt 210, the variant nucleotide is A; when the variant nucleotide is at nt 345, the variant nucleotide is G; and when the variant nucleotide is at nt 378, the variant nucleotide is C; and RNA equivalents of the nucleic acid sequences. These probes are capable of hybridizing with the HPV-68 variants shown in Figure 10.
MM4 (n = 5) and MM9 (n = 5) specimens showed a relatively large number of nucleotide substitutions; however, the sample sizes for these HPV sequences were small (Figs. 11 and 12). One MM4 specimen (23981) was classified as prototype, while the remainder had one to five nucleotide changes each. None of the MM9 specimens were identical to the reference sequence and most (n = 4) of the specimens had five or six nucleotide substitutions, two (at nt 102 and 303) that were not observed in any other specimens analyzed (Fig. 12).
Overall, it has been found that the MM4 has variations at nt 1 12, 140, 144, 249, 268, 291, and 370 of SEQ ID NO: l 1 below. Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for MM4 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 11 1 , 1 13 to 139, 145 to 248, 250 to 267, 269 to 290, 292 to 369 and 317 to 455 of SEQ ID NO:l 1 and their RNA equivalents. These probes avoid the region of variation in MM4 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for MM4 comprising oligonucleotides of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:l l which include a variant nucleotide located at at least one of numbered positions 1 12, 140, 144, 249, 268, 291 , and 370 wherein: when the variant nucleotide is at nt 1 12, the variant nucleotide is A; when the variant nucleotide is at nt 140, the variant nucleotide is G; when the variant nucleotide is at nt 144, the variant nucleotide is G or T; when the variant nucleotide is at nt 249, the variant nucleotide is C; when the variant nucleotide is at nt 268, the variant nucleotide is C; when the variant nucleotide is at nt 291, the variant nucleotide is C; and when the variant nucleotide is at nt 370, the variant nucleotide is G; and RNA equivalents of the nucleic acid sequences. These probes are capable of hybridizing with the MM4 variants shown in Figure 1 1.
Overall, it has been found that the MM9 has variations at 102, 144, 170, 187, 303, 333, 375, and 417 of SEQ ID NO:12 below. Therefore, in one prefeπed embodiment, the present invention provides nucleic acid probes for MM9 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences from numbered positions 1 to 101, 102 to 143, 145 to 169, 188 to 302, 304 to 334, 336 to 374, 376 to 416 and 418 to 458 of SEQ ID NO: 12 and their RNA equivalents. These probes avoid the region of variation in MM9 while providing a probe of a convenient size for conventional hybridization techniques.
In another prefeπed embodiment, the present invention provides nucleic acid probes for MM9 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO: 12 which include a variant nucleotide located at at least one of numbered positions 102, 144, 170, 187, 303, 333, 375, and 417 wherein: when the variant nucleotide is at nt 102, the variant nucleotide is A; when the variant nucleotide is at nt 144, the variant nucleotide is A; when the variant nucleotide is at nt 170, the variant nucleotide is A; when the variant nucleotide is at nt 187, the variant nucleotide is T; when the variant nucleotide is at nt 303, the variant nucleotide is G; when the variant nucleotide is at nt 333, the variant nucleotide is G; when the variant nucleotide is at nt 375, the variant nucleotide is T; and when the variant nucleotide is at nt 417, the variant nucleotide is A; and RNA equivalents of the nucleic acid sequences. These probes arc capable of hybridizing with the MM9 variants shown in Figure 12.
EXAMPLE 1
The present invention will now be described by means of a prophetic example. The process described in this example is suitable for creating any ofthe probes ofthe present invention. EXAMPLE
Process for developing probe for target DNA sequence of HPV
A desired sequence region of all known HPV types is aligned using a conventional nucleic acid alignment program, such as the PileUp program produced by the Wisconsin Genetics Computer Group (GCG).
Potential type-specific oligonucleotide probes are marked using the following criteria: length of 15 to 30 bp, preferably 18 to 22 bp; preferably a G/C to A/T ratio of 50:50, if possible; preferably no strings of consecutive A, T or A and T longer than 4, if possible;
T„, of approximately 58 to 62 degrees Celsius, as determined by the formula:
(# of G/C * 4°C) ÷ (# of A/T * 2°C) = Tm
One G or C is weighted as 4°C
One A or T is weighted as 2°C
G=Guanine, C=Cytosine, A=Adenosine, T=Thymine; preferably include any combination of two CG (i.e., CG, GC, GG, CC) on both ends of the probe if possible; and gross visual uniqueness of sequence as compared to other HPV type sequences at that exact location.
All potential probe sequences chosen by visual inspection are entered into any sequence entry program, such as the Scqued program of GCG.
Using any program that aligns short sequences against a selected database, the chosen probes are compared against all known HPV sequences in the selected regions. A suitable program is the FASTA program of GCG. Finally, probes having at least four nucleotide mismatches when compared with all known HPV types (except when compared to the HPV-type that the oligonucleotide probe is specific for) are chosen, with a preference for probes with greater than four mismatches.
Although probes produced by this process are designed for a predicted hybridization temperature (50 to 55°C), this procedure can be changed slightly for other hybridization parameters. For example, a lower hybridization temperature will require the oligonucleotide type- or allele-specific probe to have a greater number of mismatches and different length.
Other suitable techniques for producing HPV probes, including those of the invention, are well-known and are described in U.S. Patent No. 5,447,839 to Manos et al, U.S. Patent No. 5,182,377 to Manos et al, U.S. Patent No. 4,983,728 to Herzog et al. and U.S. Patent No. 4,849,332 to Lorincz, the entire disclosures and contents of which are hereby incoφorated by reference.
Although the present invention has been fully described in conjunction with the prefeπed embodiment thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims, unless they depart therefrom.
In the following sequences: A represents adenine; C represents cytosine; G represents guanine; T represents thymine; U represents uracil; M represents A or C; R represents A or G; W represents A or T/U; S represents C or G; Y represents C or T/U; K represents G or T/U; V represents A or C or G, not T/U; H represents A or C or T/U, not G; D represents A or G or T/U, not C; B represents C or G or T/U, not A; and N represents (A or C or G or T/U) or (unknown or other). SEQUENCE LISTING
(1) GENERAL INFORMATION:
(1) APPLICANT: Wheeler, Cosette M. Manos, Michele
(11) TITLE OF INVENTION: Probes for Detection of Human Papillomavirus
(ill) NUMBER OF SEQUENCES: 12
(iv) CORRESPONDENCE ADDRESS-
(A) ADDRESSEE: Jagtiam & Associates
(B) STREET- 6126 Rocky Way Court
(C) CITY: Centreville
(D) STATE: VA
(E) COUNTRY: USA
(F) ZIP: 20120-3400
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE. Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE-
(C) CLASSIFICATION:
(vm) ATTORNEY/AGENT INFORMATION:
(A) NAME: Jagtiam, A]ay A
(B) REGISTRATION NUMBER. 35,205
(C) REFERENCE/DOCKET NUMBER: UNME-0009-1-PCT
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (703)855-5869 (3) TELEFAX. (703)803-9387
(2) INFORMATION FOR SEQ ID Nθ:l.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 455 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:
GCACAGGGTC ATAACAATGG TRTYTGCTGG CATAATCAAT TATTTGTTAC TGTGGTAGAT 60
ACCACTCSYA GTACCAATTT AACAATATGT GCTTCTACAC AGTCTCCTGT ACCTGGGCAR 120
TATGATGCTA CCAAATTTAR GCAGTATAGC AGACATGTTG ARGAATATGA TTTGCAGTTT 180
ATTTTTCAGT TRTGTACTAT TACTTTAACT GCAGATGTTA TGTCCTATAT TCATAGTATG 240 AATAGCAGTA TTTTAGAGGA TTGGAACTTT GGTGTTCCCC CCCCSCCAAC TACTAGTTTG 300
GTGGATACAT ATCGTTTTGY ACAATCTGTT GCTATTACCT GTCAAAAGGA TGCTGCACCR 360
GCTGAAAATA AGGATCCCTA TGATAVGTTA AAGTTTTGGA ATGTGGATTT AAAGGAAAAG 420
TTTTCTTTRG ACTTAGATCA ATATCCCCTT GGACG 455 (2) INFORMATION FOR SEQ ID NO: 2 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. double
(D) TOPOLOGY, circular
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCACAAGGTC ATAATAATGG TATTTGTTGG GGCAATCAGG TATTTGTTAC TGTGGTAGAT 60
ACCACTCGCA GTACTAATAT GACTTTATGC ACACAAGTMA CTAGTGACAG TACATATAAA 120
AATGARAATT TTAAAGAATA TATAAGACAT GTTGAAGAAT ATGATYTACA GTTTGTTTTT 180
CAACTATGCA AAGTTACCTT AACTGCAGAA GTTATGACAT ATATTCATGC TATGAATCCA 240
GATATTTTAG AAGATTGGCA ATTTGGTTTA ACACCTCCTC CATCTGCTAG TTTACAGGAT 300
ACCTATAGGT TTGTTACCTC TCAGGCTATT ACGTGTCAAA AAACAGTACC TCCAAAGGAA 360
AAGGAAGACC CCTTAGGTAA ATATACATTT TGGGAAGTGR ATTTAAAGGA AAAATTTTCA 420
GCAGATTTAG ATCAGTTTCC TTTGGGACG 449 (2) INFORMATION FOR SEQ ID NO: 3 :
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 452 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY, circular
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GCACAAGGCC ATAATAATGG TATTTGTTGG AGTAACCAAT TGTTTGTTAC TGTAGTTGAT 60
ACAACCCGTA GTACAAATAT GTCTGTGTGT TCTGCTGTGT CTACTAGTGA CAGTACATAT 120
AAAAATGACA ATTTTAAGGA ATATTTAAGG CATGGTGAAG AATATGATTT ACAGTTTATT 180
TTTCAGTTAT GTAAAATAAC ACTAACAGCA GATGTTATGA CATATATTCA TAGTATGAAC 240
CCGTCCATTT TAGAGGATTG GAATTTTGGC CTTACACCAC CGCCTTCTGG TACCTTAGAG 300 /US97/06354
GACACATATC GCTATGTAAC ATCACAGGCT GTAACTTGTC AAAAACCCAG TGCACCAAAA 360
CCTAAAGATG ATCCATTAAA AAATTATACT TTTTGGGAGG TTGATTTAAA GGAAAAGTTT 420
TCTGCAGACT TAGATCAGTT TCCGTTGGGC CG 452
(2) INFORMATION FOR SEQ ID NO:4 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 455 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :
GCCCAGGGTC ATAATAATGG TATATGTTGG CATAATCAAT TATTTCTTAC TGTWGTGGAC 60
ACTACCCGTA GTACCAACTT TACATTATCT ACCTCTATAG AGTCTTCCAT ACCTTCTACA 120
TATGATCCTT CTAAGTTTAA GGAATATAYC AGGCACGTGG AGGAGTATGA TTTACAATTT 180
ATATTTCAAC TGTGTACTGT MACATTAACA ACTGATGTTA TGTCTTATAT TCACACTATG 240
AATTCCTCTA TATTGGACAA TTGGAATTYY GCTGTAGCTC CTCCACCATC TGCCAGTTTG 300
GTAGACACTT ACAGATACYT ACAGTCTGCA GCCATTACAT GTCAAAAGGA TGCTCCAGCA 360
CCTGAAAAGA AAGATCCATA TGACGGTCTA AAGTTTTGGA ATGTTGACTT AMGGGAAAAG 420
TTTAGTTTGG AACTTGATCA GTATCCCTTG GGACG 455 (2) INFORMATION FOR SEQ ID NO:5 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 455 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ll) MOLECULE TYPE. DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :
GCCCAGGGCC ATAACAATGG TATTTGTTGG CATAATCAGT TGTTTGTTAC TGTAGTGGAY 60
ACTACCCGCA GTACTAATTT AACATTATGT GCCTCTACAS AAAMTCCTGT GCCARRTACA 120
TATGAYCCTA CTAAGTTTAA GCAVTATAGT AGACATGTGG AGGAATATGA TTTACAGTTT 180
ATTTTTCAGT TGTGCACTAT TACTTTAACT GCAGAGGTTA TGTCATATAT CCATAGTATG 240
AATAGTAGTA TATTYGAAAA TTGGAATTTT GGTGTRCCTC CACCACCTAC YACAAGTTTR 300
RTGGATACAT ATCGTTTTGT GCAATCAGTT GCTGTTACCT GTCAAAAGRA TARTACACCT 360 CCAGAAAAGC AGGATCCATA TGATAAATTR AAGTTTTGGA CTGTTGACCT AAAGGAAAAA 420 TTTTCCTCCG ATTTRGATCA ATATCCCCTT GGTCG 455
(2) INFORMATION FOR SEQ ID NO:6 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 452 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCGCAGGGTC ACAATAATGG CATTTGCTGG AACAATCAGC TTTTTATTAC CTGTGTTGAT 60
ACTACCAGAA GTACAAATTT AACTATTAGC ACTGCCACTG CTGCGGTTTC CCCAACATTT 120
ACTCCAAGTA ACTTTAAGCA ATATATTAGG CATGGGGAAG AGTATGAATT GCAATTTATT 180
TTTCAATTAT GTAAAATTAC TTTAACTACA GAGGTAATGG CTTATTTACA CACAATGGAT 240
CCTACCATTC TTGAACAGTG GAATTTTGGA TTAACATTAC CTCCGTCTGC TAGTTTGGAG 300
GATGCATATA GGTTTGTTAG AAATGCAGCT ACTAGCTGTC AAAAGGATAC CCCTCCACAG 360
GCTAAGCCAG ATCCTTTGGC CAAATATAAA TTTTGGGATG TTGATTTAAA GGAACGATTT 420
TCTTTAGATT TAGACCAATT TGCATTGGGT CG 452 (2) INFORMATION FOR SEQ ID NO:7:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY, circular
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GCGCAGGGCC ACAATAATGG CATATGTTGG GGCAATCAGT TGTTTGTCAC AGTTGTGGAT 60
ACCACTCGTA GCACTAACAT GACTTTATGT GCTGARGTKA MAAAGGAARR CACATATAAA 120
AATGAAAATT TTAAGGAATA CCTTCGTCAT GGCGAGGAAT TYGATTTACA ATTTATTTTT 180
CAATTGTGCA ARATTACATT AACAGCTGAT GTTATGACAT AYATTCATAA RATGGATGCC 240
ACTATYTTAG AGGACTGGCA ATTTGGCCTT ACCCCACCAC CGTCTGCATC TTTGGAGGAC 300
ACATACAGAT TTGTMACKTC TACTGCTATA ACWTGTCARA ARAACACACC ACCTAARGGA 360
AAGGAAGATC CTTTAAARGA STATATGTTY TGGGAGGTGG ATTTAAAAGA AAAGTTTTCT 420 GCAGATTTAG ATCAGTTTCC TTTAGGTAG 449
(2) INFORMATION FOR SEQ ID NO:8 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ll) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 :
GCACAAGGTC ATAACAATGG CATTTGCTGG GGCAATCAGT TATTTGTTAC CGTRGTTGAT 60
ACCACTCGTA GCACTAATAT GACATTATGC ACTGAAGTAA CTAARGAAGR TACATATAAA 120
AATRATAATT TTAAGGAATA TGTACGTCAT GTTGAAGAAT ATGACTTACA GTTTGTTTTT 180
CAGCTTTGCA AAATTACACT AACTGCAGAG RTAATGACAT ATATACATAC TATGRATTCM 240
RATATTTTGG AGGACTGGCA ATTTGGTTTA ACACCTCCTC CGTCTGCCAG TTTRCAGGAC 300
ACATATAGAT TTGTTACCTC CCAGGCTATT ACTTGCCAAA AAACAGCACC CCCTAAAGAA 360
AAGGAAGATC CATTAAATAA ATATACTTTT TGGGAGGTTA ACTTAAAGGA AAAGTTTTCT 420
GCAGATCTRG ATCAGTTTCC TTTGGGACG 449 (2) INFORMATION FOR SEQ ID NO:9:
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 455 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE. DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9■
GCTCAGGGTT TAAACAATGG TATATGTTGG CACAATCAAT TGTTTTTAAC AGTTGTAGAT 60
ACTACYCGCA GCACCAATCT TTCTGTGTGT GCTTCTACTA CKBCTTCTAT TCCTAATGTA 120
TACACACCTA CCAGTTTTAA AGAATATGCC AGACATGTGG AGGAATTTGA TTTGCAGTTT 180
ATATTTCAAC TGTGTAAAAT AACATTAACT ACAGAGGTAA TGTCATACAT TCATAATATG 240
AATACCACTA TTTTGGAGGA TTGGAATTTT GGTGTTACAC CACCTCCTAC TGCTAGTTTA 300
GTTGACACAT ACCGTTTTGT TCAATCTGCT GCTGTAACTT GTCAAAAGGA CACCGCACCG 360
CCAGTTAAAC AGGACCCTTA TGACAAACTA AAGTTTTGGM CTGTAGATCT TAAGGAAAGG 420
TTTTCTGCAG ATCTTGATCA GTTTCCTTTG GGACG 455 INFORMATION FOR SEQ ID NO 10
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 455 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS double
(D) TOPOLOGY circular
(n) MOLECULE TYPE DNA (genomic)
(xi) SEQUENCE DESCRIPTION SEQ ID NO 10
GCACAGGGAC ACAACAATGG TATTTGTTGG CATAATCAAT TATTTCTTAC TGTTGTGGAT 60
ACCACTCGCA GTACCAATTT TACTTTGTCT ACTACTACTG AATCAGCTGT ACCAAATATT 120
TATGATCCTA ATAAATTTAA GGAATATATT AGGCATGTTG AGGAATATGA TTTGCAATTT 180
ATATTTCAGT TGTGTACTAT AACATTGTCM ACTGATGTAA TGTCCTATAT ACATACTATG 240
AATCCTGCTA TTTTGGATGA TTGGAATTTT GGTGTTGCCC CTCCACCATC TGCTAGTCTT 300
GTAGATACAT ACCGCTATCT GCAATCAGCA GCAATTACAT GTCARAAAGA CGCCCCTGCA 360
CCTACTAAAA AGGATCCMTA TGATGGCTTA AACTTTTGGA ATGTAAATTT AAAGGAAAAG 420
TTTAGTTCTG AACTGGACCA GTTTCCTTTA GGACG 455 (2) INFORMATION TOR SEQ ID NO 11
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 455 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS double
(D) TOPOLOGY circular
(ii) MOLECULE TYPE DNA (genomic)
(xi) SEQUENCE DESCRIPTION SEQ ID NO 11
GCACAGGGAC ATAATAATGG CATTTGCTGG AATAATCAGC TTTTTATTAC TTGTGTTGAC 60
ACTACTAGAA GTACCAATTT AACCATTAGC ACTGCTGTTA CTCAATCTGT TRCACAAACA 120
TTTACTCCAG CAAACTTTAR GCADTACATT AGGCATGGGG AAGAATATGA ATTGCAATTT 180
ATATTTCAAT TGTGTAAAAT CACTTTAACT ACTGAAATTA TGGCTTACCT GCACACCATG 240
GATTCTACMA TTTTAGAACA GTGGAATYTT GGATTAACCT TGCCCCCCTC MGCTAGTTTG 300
GAGGATGCCT ATCGATTTGT AAAAAATGCA GCAACATCCT GTCACAAGGA CAGTCCTCCA 360
CAGGCTAAAS AAGACCCTTT GGCAAAATAT AAATTTTGGA ATGTAGACCT TAAGGAACGC 420
TTTTCTTTGG ATTTGGATCA GTTTCCTTTT GGACG 455 (2) INFORMATION FOR SEQ ID NO 12 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 458 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GCACAGGGTC ATAATAATGG TATTTGTTGG CATAATCAAT TATTTTTAAC TGTTGTAGAT 60
ACTACTAGAA GCACTAATTT TTCTGTATGT GTAGGTACAC ARGCTAGTAG CTCTACTACA 120
ACGTATGCCA ACTCTAATTT TAARGAATAT TTAAGACATG CAGAAGAGTW TGATTTACAG 180
TTTGTTYTTC AGTTATGTAA AATTAGTTTA ACTACTGAGG TAATGACATA TATACATTCT 240
ATGAATTCTA CTATATTGGA AGAGTGGAAT TTTGGTCTTA CCCCACCACC GTCAGGTACT 300
TTRGAGGAAA CATATAGATA TGTAACATCA CAKGCTATTA GTTGCCAACG TCCTCAACCT 360
CCTAAAGAAA CAGAKGACCC ATATGCCAAG CTATCCTTTT GGGATGTAGA TCTTAARGAA 420
AAGTTTTCTG CAGAATTAGA TCAGTATCCC CTTGGACG 458

Claims

WHAT IS CLAIMED
1. A nucleic acid probe for HPV-18 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 21 , 25 to 67, 70 to 1 19, 121 to 139, 163 to 191 , 193 to 284, 286 to 319, 321 to 359, 361 to 385, 387 to 428 and 430 to 455 of SEQ ID NO: l and their RNA equivalents.
2. A nucleic acid probe for HPV-33 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 98, 100 to 125, 127 to 165, 167 to 399 and 401 to 449 of SEQ ID NO:2 and their RNA equivalents.
3. A nucleic acid probe for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 102 and 104 to 452 of SEQ ID NO:3 and their RNA equivalents.
4. A nucleic acid probe for HPV-39 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 53, 55 to 148, 150 to 200, 202 to 268, 271 to 318, 320 to 41 1 and 413 to 455 of SEQ ID NO:4 and their RNA equivalents.
5. A nucleic acid probe for HPV-45 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 59, 61 to 99, 145 to 254, 256 to 275, 302 to 348, 354 to 389, 391 to 434 and 436 to 455 of SEQ ID NO:5 and their RNA equivalents.
6. A nucleic acid probe for HPV-51 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 347 and 349 to 452 of SEQ ID NO:6 and their RNA equivalents.
7. A nucleic acid probe for HPV-52 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 95, 111 to 161, 163 to 191, 193 to 221, 247 to 314, 358 to 377 and 391 to 449 of SEQ ID NO:7 and their RNA equivalents.
8. A nucleic acid probe for HPV-58 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 53, 55 to 104, 125 to 210, 212 to 234, 242 to 293, 295 to 428 and 430 to 449 of SEQ ID NO:8 and their RNA equivalents.
9. A nucleic acid probe for HPV-59 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 65, 67 to 101, 104 to 399 and 401 to 455 of SEQ ID NO:9 and their RNA equivalents.
10. A nucleic acid probe for HPV-68 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 209, 1 1 1 to 344, 346 to 377 and 379 to 455 of SEQ ID NO: 10 and their RNA equivalents.
11. A nucleic acid probe for HPV-MM4 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 11 1 , 1 13 to 139, 145 to 248, 250 to 267, 269 to 290, 292 to 369 and 317 to 455 of SEQ ID NO:l 1 and their RNA equivalents.
12. A nucleic acid probe for HPV-MM9 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences from numbered positions 1 to 101, 102 to 143, 145 to 169, 188 to 302, 304 to 334, 336 to 374, 376 to 416 and 418 to 458 of SEQ ID NO.12 and their RNA equivalents.
13. A nucleic acid probe for HPV-18 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO: l which a variant nucleotide at at least one of numbered positions 22, 24, 68, 69, 120, 140, 162, 192, 285, 320, 360, 386, and 429 wherein: when said variant nucleotide is at nt 22, said variant nucleotide is A; when said variant nucleotide is at nt 24, said variant nucleotide is C; when said variant nucleotide is at nt 68, said variant nucleotide is G; when said variant nucleotide is at nt 69, said variant nucleotide is T or U; when said variant nucleotide is at nt 120, said variant nucleotide is G; when said variant nucleotide is at nt 140, said variant nucleotide is G; when said variant nucleotide is at nt 162, said variant nucleotide is A; when said variant nucleotide is at nt 192, said variant nucleotide is A; when said variant nucleotide is at nt 285, said variant nucleotide is G; when said variant nucleotide is at nt 320, said variant nucleotide is C; when said variant nucleotide is at nt 386, said variant nucleotide is C or G; and when said variant nucleotide is at nt 32, said variant nucleotide is G; and RNA equivalents of said nucleic acid sequences.
14. A nucleic acid probe for HPV-33 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:2 which include a variant nucleotide at at least one of numbered positions 99, 126, 166, and 400 wherein: when said variant nucleotide is at nt 99, said variant nucleotide is C; when said variant nucleotide is at nt 126, said variant nucleotide is G; when said variant nucleotide is at nt 166, said variant nucleotide is T; and when said variant nucleotide is at nt 400, said variant nucleotide is A; and RNA equivalents of said nucleic acid sequences.
15. A nucleic acid probe for HPV-35 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:3 which include nt 103.
16. A nucleic acid probe for HPV-39 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:4 which include a variant nucleotide at at least one of numbered positions 54, 149, 201, 269, 270, 319 and 412 wherein: when said variant nucleotide is at nt 54, said variant nucleotide is A; when said variant nucleotide is at nt 149, said variant nucleotide is T; when said variant nucleotide is at nt 201, said variant nucleotide is A; when said variant nucleotide is at nt 269, said variant nucleotide is C; when said variant nucleotide is at nt 270, said variant nucleotide is C; when said variant nucleotide is at nt 319, said variant nucleotide is T or U; and when said variant nucleotide is at nt 412, said variant nucleotide is C; and RNA equivalents of said nucleic acid sequences.
17. A nucleic acid probe for HPV-45 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO: 5 which include a variant nucleotide at at least one of numbered positions 60, 100, 104, 1 15, 1 16, 126, 144, 255, 276, 291 , 300, 301, 349, 353, 390, and 435 wherein: when said variant nucleotide is at nt 60, said variant nucleotide is T; when said variant nucleotide is at nt 100, said variant nucleotide is G; when said variant nucleotide is at nt 104, said variant nucleotide is C; when said variant nucleotide is at nt 1 15, said variant nucleotide is G; when said variant nucleotide is at nt 1 16, said variant nucleotide is A; when said variant nucleotide is at nt 126, said variant nucleotide is T or U; when said variant nucleotide is at nt 144, said variant nucleotide is A or C; when said variant nucleotide is at nt 255, said variant nucleotide is G; when said variant nucleotide is at nt 276, said variant nucleotide is A or G; when said variant nucleotide is at nt 291, said variant nucleotide is C; when said variant nucleotide is at nt 300, said variant nucleotide is A; when said variant nucleotide is at nt 301, said variant nucleotide is A; when said variant nucleotide is at nt 349, said variant nucleotide is A; when said variant nucleotide is at nt 353, said variant nucleotide is G; when said variant nucleotide is at nt 390, said variant nucleotide is G; and when said variant nucleotide is at nt 435, said variant nucleotide is A; and RNA equivalents of said nucleic acid sequences.
18. A nucleic acid probe for HPV-51 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:6 which include nt 348.
19. A nucleic acid probe for HPV-52 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO: 7 which include a variant nucleotide at at least one of numbered positions 96, 99, 101, 109, 1 10, 162, 192, 222, 231, 246, 315, 318, 333, 339, 342, 357, 378, 381, and 390 wherein: when said variant nucleotide is at nt 96, said variant nucleotide is A; when said variant nucleotide is at nt 99, said variant nucleotide is G; when said variant nucleotide is at nt 101, said variant nucleotide is C; when said variant nucleotide is at nt 109, said variant nucleotide is G; when said variant nucleotide is at nt 1 10, said variant nucleotide is A; when said variant nucleotide is at nt 162, said variant nucleotide is C; when said variant nucleotide is at nt 192, said variant nucleotide is G; when said variant nucleotide is at nt 222, said variant nucleotide is T; when said variant nucleotide is at nt 231 , said variant nucleotide is A; when said variant nucleotide is at nt 246, said variant nucleotide is C; when said variant nucleotide is at nt 315, said variant nucleotide is A; when said variant nucleotide is at nt 318, said variant nucleotide is G; when said variant nucleotide is at nt 333, said variant nucleotide is A; when said variant nucleotide is at nt 339, said variant nucleotide is G; when said variant nucleotide is at nt 342, said variant nucleotide is G; when said variant nucleotide is at nt 357, said variant nucleotide is G; when said variant nucleotide is at nt 378, said variant nucleotide is A; when said variant nucleotide is at nt 381, said variant nucleotide is G; and when said variant nucleotide is at nt 390, said variant nucleotide is C; and RNA equivalents of said nucleic acid sequences.
20. A nucleic acid probe for HPV-58 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:8 which include a variant nucleotide at at least one of numbered positions 54, 105, 110, 124, 211, 235, 240, 241, 294, and 429 wherein: when said variant nucleotide is at nt 54, said variant nucleotide is A; when said variant nucleotide is at nt 105, said variant nucleotide is A; when said variant nucleotide is at nt 1 10, said variant nucleotide is A; when said variant nucleotide is at nt 124, said variant nucleotide is A; when said variant nucleotide is at nt 21 1, said variant nucleotide is G; when said variant nucleotide is at nt 235, said variant nucleotide is A; when said variant nucleotide is at nt 240, said variant nucleotide is A; when said variant nucleotide is at nt 241, said variant nucleotide is G; when said variant nucleotide is at nt 294, said variant nucleotide is G; and when said variant nucleotide is at nt 429, said variant nucleotide is G; and RNA equivalents of said nucleic acid sequences.
21. A nucleic acid probe for HPV-59 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO:9 which include a variant nucleotide at at least one of numbered positions 66, 102, 103, and 400 wherein: when said variant nucleotide is at nt 66, said variant nucleotide is C; when said variant nucleotide is at nt 102, said variant nucleotide is G; when said variant nucleotide is at nt 103, said variant nucleotide is C or G; and when said variant nucleotide is at nt 400, said variant nucleotide is A; and RNA equivalents of said nucleic acid sequences.
22. A nucleic acid probe for HPV-68 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO: 10 which include a variant nucleotide at at least one of numbered positions 210, 345 and 378 wherein: when said variant nucleotide is at nt 210, said variant nucleotide is A; when said variant nucleotide is at nt 345, said variant nucleotide is G; and when said variant nucleotide is at nt 378, said variant nucleotide is C; and RNA equivalents of said nucleic acid sequences.
23. A nucleic acid probe for MM4 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting of the nucleic acid sequences contained within SEQ ID NO:l 1 which include a variant nucleotide at at least one of numbered positions 112, 140, 144, 249, 268, 291 , and 370 wherein: when said variant nucleotide is at nt 112, said variant nucleotide is A; when said variant nucleotide is at nt 140, said variant nucleotide is G; when said variant nucleotide is at nt 144, said variant nucleotide is G or T; when said variant nucleotide is at nt 249, said variant nucleotide is C; when said variant nucleotide is at nt 268, said variant nucleotide is C; when said variant nucleotide is at nt 291, said variant nucleotide is C; and when said variant nucleotide is at nt 370, said variant nucleotide is G; and RNA equivalents of said nucleic acid sequences.
24. A nucleic acid probe for MM9 comprising an oligonucleotide of 15 to 30 nucleotides selected from the group consisting ofthe nucleic acid sequences contained within SEQ ID NO: 12 which include a variant nucleotide at at least one of numbered positions 102, 144, 170, 187, 303, 333, 375, and 417 wherein: when said variant nucleotide is at nt 102, said variant nucleotide is A; when said variant nucleotide is at nt 144, said variant nucleotide is A; when said variant nucleotide is at nt 170, said variant nucleotide is A; when said variant nucleotide is at nt 187, said variant nucleotide is T; when said variant nucleotide is at nt 303, said variant nucleotide is G; when said variant nucleotide is at nt 333, said variant nucleotide is G; when said variant nucleotide is at nt 375, said variant nucleotide is T; and when said variant nucleotide is at nt 417, said variant nucleotide is A; and RNA equivalents of said nucleic acid sequences.
PCT/US1997/006354 1996-04-15 1997-04-14 Probes for the detection of human papillomavirus WO1997039010A1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1082466A4 (en) * 1998-05-30 2004-10-06 Bayer Healthcare Llc Method, reagent and kit for genotyping of human papillomavirus
WO2006077102A3 (en) * 2005-01-18 2007-07-19 Delft Diagnostic Lab Bv Method for the detection of hpv and probes, primers and kits
US20120009562A1 (en) * 2004-05-07 2012-01-12 Roche Molecular Systems, Inc. High-Risk Human Papillomavirus Detection
US20130078618A1 (en) * 2004-12-08 2013-03-28 Gen-Probe Incorporated Compositions, reaction mixtures and methods for detecting nucleic acids from type a1 and/or type c1 human papillomavirus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182377A (en) * 1988-09-09 1993-01-26 Hoffmann-La Roche Inc. Probes for detection of human papillomavirus
US5447839A (en) * 1988-09-09 1995-09-05 Hoffmann-La Roche Inc. Detection of human papillomavirus by the polymerase chain reaction
US5527898A (en) * 1988-09-09 1996-06-18 Hoffmann-La Roche Inc. Detection of human papillomavirus by the polymerase chain reaction
US5643715A (en) * 1988-10-26 1997-07-01 Lancaster; Wayne D. Human papillomavirus type 52 DNA sequences and methods for employing the same

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6352825B1 (en) * 1994-02-21 2002-03-05 Stichting Researchfonds Pathologie Human Papilloma Virus detection in a nucleic acid amplification process using general primers

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182377A (en) * 1988-09-09 1993-01-26 Hoffmann-La Roche Inc. Probes for detection of human papillomavirus
US5447839A (en) * 1988-09-09 1995-09-05 Hoffmann-La Roche Inc. Detection of human papillomavirus by the polymerase chain reaction
US5527898A (en) * 1988-09-09 1996-06-18 Hoffmann-La Roche Inc. Detection of human papillomavirus by the polymerase chain reaction
US5643715A (en) * 1988-10-26 1997-07-01 Lancaster; Wayne D. Human papillomavirus type 52 DNA sequences and methods for employing the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1007538A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1082466A4 (en) * 1998-05-30 2004-10-06 Bayer Healthcare Llc Method, reagent and kit for genotyping of human papillomavirus
US20120009562A1 (en) * 2004-05-07 2012-01-12 Roche Molecular Systems, Inc. High-Risk Human Papillomavirus Detection
US8202975B2 (en) * 2004-05-07 2012-06-19 Roche Molecular Systems, Inc. High-risk human papillomavirus detection
US20130078618A1 (en) * 2004-12-08 2013-03-28 Gen-Probe Incorporated Compositions, reaction mixtures and methods for detecting nucleic acids from type a1 and/or type c1 human papillomavirus
US8574841B2 (en) * 2004-12-08 2013-11-05 Gen-Probe Incorporated Compositions, reaction mixtures and methods for detecting nucleic acids from type A1 and/or type C1 human papillomavirus
WO2006077102A3 (en) * 2005-01-18 2007-07-19 Delft Diagnostic Lab Bv Method for the detection of hpv and probes, primers and kits

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