FIELD OF THE INVENTION
The present invention relates to a method and a detector for detecting human papilloma viruses, and more particularly to a method and a detector for simultaneously detecting and identifying subtype of human papilloma viruses (HPV).
BACKGROUND OF THE INVENTION
In humans, more than 70 genetically distinct strains of human papilloma virus (HPV) have been identified based on DNA hybridization studies. According to some reports, different HPV types cause distinct diseases. For example, “Low-risk” HPVs, e.g., HPV 6 and HPV 11, cause benign hyperplasias such as genital warts, while “high-risk” HPVs, e.g., HPV-16, HPV-18, HPV-31, HPV-33, HPV-54, and the like, can cause cancers such as cervical or penile carcinoma.
Cervical cancer is the most common cancer in women. The consorts are often men with penile warts. Sexual activity appears to be an important predisposing factor of the epidemic disease and precancerous lesions. In early 5 to 10 years during the development of cervical cancer, cervical cells form cervical intraepithelial neoplasm.
Recently, in order to decrease the incidence of cervical cancer, Pap smear is used for the cervical cancer screening. However, the Pap smear has a false negative rate of about 30%˜40%. In addition, it is known that more that 95% of cervical carcinoma tissue contain detectable DNA sequences for known varieties of the human papilloma virus (HPV). Hence, the combination of Pap smear and HPV detection for the cervical cancer screening is necessarily considered.
The Applicant cooperates with the hospital to do the epidemiological research in women cervical cancer by using Pap smear and HPV detection, wherein the HPV detection is proceeded by using polymerase chain reaction and nucleotide sequencing. There are 2424 women aged from 16 to 84 for the epidemiology research, wherein 1963 women provide the effective specimen. The research results are shown as follows.
- 1) 1.9% (37/1963) of the women have abnormal cytological smears.
- 2) 12.7% (244/1926) of the women with normal cytological smears but have HPV infection.
- 3) The HPV prevalence in the women with abnormal cytological smears is 51.4% (19/37) and positively relative to the degree of the abnormal cytological smears, wherein the incidence of abnormal non-typical squamous cells is 23.1%, the incidence of low abnormal epithelial cells is 41.7%, and the incidence of high abnormal epithelial cells is 75%.
- 4) The subtypes of human papilloma viruses detected in the specimens are HPV 52, HPV 58, HPV 70, HPV 16, HPV 18, HPV 68, HPV 33, HPV 66, HPV 35, HPV 37, HPV 54, HPV 59, HPV 67, HPV 72, HPV 69, HPV 82, HPV 39, HPV 31, HPV 32, HPV HLT7474-S, HPV 6, HPV CP8061, HPV 62, HPV CP8304, HPV 44, HPV 11, HPV 61, HPV 74, HPV 42 and HPV 43.
The conventional HPV detecting kits are only used for detecting 18 subtypes of human papilloma viruses including high risk HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59 and HPV 68, and detecting low risk HPV 6, HPV I1, HPV 42, HPV 43 and HPV 44.
However, according to the comparison of the epidemiology research and the conventional HPV detecting kits, several clinically-important subtypes of human papilloma viruses contained in a specimen could not be identified by the conventional HPV detecting kits. In addition, the conventional HPV detecting kits only tell the information of HPVs contained in a specimen by two categories, high risk HPVs or low HPVs, rather than tell the definite subtypes as which they are classified. Therefore, except the high risk HPVs and the low risk HPVs, if other HPV subtypes are contained in the specimen, the conventional HPV detecting kits can not identify immediately, which would seriously affects the diagnosis accuracy. Furthermore, the conventional HPV detecting kits lack the system control for checking the house-keeping genes contained in a specimen. Without the system control, it will be hard to confirm whether the detecting protocols are precisely followed. That is, the user can not tell the positive/negative result comes from the HPV subtypes presence/absence or comes from the incorrect protocols execution. Therefore, the conventional detecting kit without the system control would not be able to provide a convincing result.
From the above description, it is known that the conventional detecting kit can not identify many HPV subtypes at the same time and it does not include an internal control in the detecting system. Therefore, how to simultaneously detect many HPV subtypes contained in a biological simple and design an accurate internal control in the detecting kits have become a major problem waited to be solved. In order to overcome the foresaid drawbacks of the conventional HPV detecting kits, the present invention provides a method and a detector for simultaneously detecting and identifying subtypes of human papilloma viruses contained in a sample.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a detector for simultaneously detecting and identifying subtypes of human papilloma viruses (HPV) contained in a sample.
The main purpose of the present invention is to provide a HPV detecting kit, which is able to diagnose multiple HPV subtypes (up to 39 different subtypes) at the same time, allowing the rapid and reliable detection and identification of HPV possibly present in a biological sample.
It is another object of the present invention to provide a rapid and reliable method to detect and identify the HPV present in a biological sample.
It is another object of the present invention to provide a HPV detecting kit with high specificity and accuracy, which includes an internal control to show whether the detecting process is well handled so that the detecting result is dependable.
It is another object of the present invention to provide a number of oligonucleotides as probes for detecting and identifying the HPV present in a biological sample.
According to one aspect of the present invention, a detector for detecting and simultaneously diagnosing at least one subtype of human papilloma viruses (HPV) contained in a biological sample, comprises: a carrier, a plurality of micro-dots immobilized on the carrier, wherein each micro-dot is for identifying one particular HPV subtype, and the HPV subtype is one selected from a group consisting of (HPV 6, HPV 1, HPV 16, HPV 18, HPV 26, HPV 31, HPV 32, HPV 33, HPV 35, HPV 37, HPV 39, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 62, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 72, HPV 74, HPV 82, HPV CP8061, HPV CP8034, HPV LIAE5, HPV MM4, HPV MM7 and HPV MM8); and at least one oligonucleotide sequence contained in each the micro-dot that is specific to the one particular HPV subtype, wherein the at least one oligonucleotide sequence serves as a detection probe that hybridizes specifically with an LI gene sequence of the one particular HPV subtype to form a hybridization complex as a detection indicator, so that each micro-dot identifies one particular HPV subtype via a corresponding oligonucleotide of the one particular HPV subtype, and thereby detecting and simultaneously identifying subtypes of human papilloma viruses.
In accordance with the present invention, the at least one oligonucleotide that hybridizes specifically with an LI gene sequence of the one particular HPV subtype is respectively chosen from the following list for each HPV subtype: (SEQ ID NO: 1-SEQ ID NO: 12) for HPV 6, (SEQ ID NO: 13-SEQ ID NO:24) for HPV 11, (SEQ ID NO:25-SEQ ID NO:36) for HPV 16, (SEQ ID NO:37-SEQ ID NO:48) for HPV 18, (SEQ ID NO:49-SEQ ID NO:58) for HPV 26, (SEQ ID NO:59-SEQ ID NO:68) for HPV 31, (SEQ ID NO:69-SEQ ID NO:79) for HPV 32, (SEQ ID NO:80-SEQ ID NO:90) for HPV 33, (SEQ ID NO:91-SEQ ID NO:100) for HPV 35, (SEQ ID NO:101-SEQ ID NO:1 12) for HPV 37, (SEQ ID NO:113-SEQ ID NO:123) for HPV 39, (SEQ ID NO: 124-SEQ ID NO: 133) for HPV 42, (SEQ ID NO: 134-SEQ ID NO: 143) for HPV 43, (SEQ ID NO: 144-SEQ ID NO: 154) for HPV 44, (SEQ ID NO: 155-SEQ ID NO: 165) for HPV 45, (SEQ ID NO: 166-SEQ ID NO: 177) for HPV 51, (SEQ ID NO:178-SEQ ID NO:189) for HPV 52, (SEQ ID NO:190-SEQ ID NO:199) for HPV 53, (SEQ ID NO:200-SEQ ID NO:209) for HPV 54, (SEQ ID NO:210-SEQ ID NO:218) for HPV 55, (SEQ ID NO:219-SEQ ID NO:228) for HPV 56, (SEQ ID NO:229-SEQ ID NO:239) for HPV 58, (SEQ ID NO:240-SEQ ID NO:250) for HPV 59, (SEQ ID NO:251-SEQ ID NO:261) for HPV 61, (SEQ ID NO:262-SEQ ID NO:272) for HPV 62, (SEQ ID NO:273-SEQ ID NO:283) for HPV 66, (SEQ ID NO:284-SEQ ID NO:294) for HPV 67, (SEQ ID NO:295-SEQ ID NO:305) for HPV 68, (SEQ ID NO:306-SEQ ID NO:316) for HPV 69, (SEQ ID NO:317-SEQ ID NO:328) for HPV 70, (SEQ ID NO:329-SEQ ID NO:341) for HPV 72, (SEQ ID NO:342-SEQ ID NO:353) for HPV 74, (SEQ ID NO:354-SEQ ID NO:362) for HPV 82, (SEQ ID NO:363-SEQ ID NO:374) for HPV CP8061, (SEQ ID NO:375-SEQ ID NO:386) for HPV CP8034, (SEQ ID NO:387-SEQ ID NO:397) for HPV L1AE5, (SEQ ID NO:398-SEQ ID NO:408) for HPV MM4, (SEQ ID NO:409-SEQ ID NO:419) for HPV MM7, and (SEQ ID NO:420-SEQ ID NO:429) for HPV MM8.
Preferably, the carrier is a nylon membrane.
Preferably, the carrier is a glass plate.
Preferably, the detector is an oligonucleotide biochip.
Preferably, the at least one oligonucleotide has a length between 15-30 bases.
Preferably, the detector further comprises a micro-dot containing a Glutaldehyde-3-phosphodehydrogenase (GAPDH) gene, which is used as an internal control.
According to another aspect of the present invention, a method for detecting and simultaneously diagnosing at least one subtype of human papilloma viruses (HPV) contained in a biological sample is provided. The detecting method comprises steps of: amplifying an L1 gene fragment of human papilloma viruses (HPV) contained in the biological sample and obtaining an amplification product by polymerase chain reaction (PCR) using primers labeled with signaling substance; hybridizing the amplification product with a detector according to claim 1 to form a hybridization complex; removing nonhybridized the amplification product; and detecting the hybridization complex through detecting the signaling substance, thereby detecting and simultaneously identifying HPV subtypes contained in the biological sample.
Preferably, the amplification product has a length of 450 base pairs by using MY09 as sense primer and MY11 as anti-sense primer in polymerase chain reaction (PCR).
Preferably, the amplification product has a length of 190 base pairs by using MY11 as sense primer and GP6+ as anti-sense primer in polymerase chain reaction (PCR).
Preferably, the signaling substance is biotin.
Preferably, the biotin reacts with avidin-alkalinephosphatase to show the hybridization result by presenting a particular color.
Preferably, the signaling substance is a fluorescent substance.
Preferably, the fluorescent substance is Cyanine 5.
According to another aspect of the present invention, a probe which hybridizes to nucleic acid from an HPV subtype, the probe being selected from the group consisting of: SEQ ID NO:1-SEQ ID NO:12 and sequences fully complementary thereto, which hybridize with HPV 6; SEQ ID NO:13-SEQ ID NO:24 and sequences fully complementary thereto, which hybridize with HPV 11; SEQ ID NO:25-SEQ ID NO:36 and sequences fully complementary thereto, which hybridize with HPV 16; SEQ ID NO:37-SEQ ID NO:48 and sequences fully complementary thereto, which hybridize with HPV 18; SEQ ID NO:49-SEQ ID NO:58 and sequences fully complementary thereto, which hybridize with HPV 26; SEQ ID NO:59-SEQ ID NO:68 and sequences fully complementary thereto, which hybridize with HPV 31; SEQ ID NO:69-SEQ ID NO:79 and sequences fully complementary thereto, which hybridize with HPV 32; SEQ ID NO:80-SEQ ID NO:90 and sequences fully complementary thereto, which hybridize with HPV 33; SEQ ID NO:91-SEQ ID NO:100 and sequences fully complementary thereto, which hybridize with HPV 35; SEQ ID NO:101-SEQ ID NO:112 and sequences fully complementary thereto, which hybridize with HPV 37; SEQ ID NO:113-SEQ ID NO: 123 and sequences fully complementary thereto, which hybridize with HPV 39; SEQ ID NO: 124-SEQ ID NO:133 and sequences fully complementary thereto, which hybridize with HPV 42; SEQ ID NO:1 34-SEQ ID NO:143 and sequences fully complementary thereto, which hybridize with HPV 43; SEQ ID NO:144-SEQ ID NO:154 and sequences fully complementary thereto, which hybridize with HPV 44; SEQ ID NO:155-SEQ ID NO:165 and sequences fully complementary thereto, which hybridize with HPV 45; SEQ ID NO:166-SEQ ID NO:177 and sequences fully complementary thereto, which hybridize with HPV 51; SEQ ID NO:178-SEQ ID NO:189 and sequences fully complementary thereto, which hybridize with HPV 52; SEQ ID NO:190-SEQ ID NO:199 and sequences fully complementary thereto, which hybridize with HPV 53; SEQ ID NO:200-SEQ ID NO:209 and sequences fully complementary thereto, which hybridize with HPV 54; SEQ ID NO:210-SEQ ID NO:218 and sequences fully complementary thereto, which hybridize with HPV 55; SEQ ID NO:219-SEQ ID NO:228 and sequences fully complementary thereto, which hybridize with HPV 56; SEQ ID NO:229-SEQ ID NO:239 and sequences fully complementary thereto, which hybridize with HPV 58; SEQ ID NO:240-SEQ ID NO:250 and sequences fully complementary thereto, which hybridize with HPV 59; SEQ ID NO:251-SEQ ID NO:261 and sequences fully complementary thereto, which hybridize with HPV 61; SEQ ID NO:262-SEQ ID NO:272 and sequences fully complementary thereto, which hybridize with HPV 62; SEQ ID NO:273-SEQ ID NO:283 and sequences fully complementary thereto, which hybridize with HPV 66; SEQ ID NO:284-SEQ ID NO:294 and sequences fully complementary thereto, which hybridize with HPV 67; SEQ ID NO:295-SEQ ID NO:305 and sequences fully complementary thereto, which hybridize with HPV 68; SEQ ID NO:306-SEQ ID NO:316 and sequences fully complementary thereto, which hybridize with HPV 69; SEQ ID NO:317-SEQ ID NO:328 and sequences fully complementary thereto, which hybridize with HPV 70; SEQ ID NO:329-SEQ ID NO:341and sequences fully complementary thereto, which hybridize with HPV 72; SEQ ID NO:342-SEQ ID NO:353 and sequences fully complementary thereto, which hybridize with HPV 74; SEQ ID NO:354-SEQ ID NO:362 and sequences fully complementary thereto, which hybridize with HPV 82; SEQ ID NO:363-SEQ ID NO:374 and sequences fully complementary thereto, which hybridize with HPV CP8061; SEQ ID NO:375-SEQ ID NO:386 and sequences fully complementary thereto, which hybridize with HPV CP8034; SEQ ID NO:387-SEQ ID NO:397 and sequences fully complementary thereto, which hybridize with HPV L1AE5; SEQ ID NO:398-SEQ ID NO:408 and sequences fully complementary thereto, which hybridize with HPV MM4; SEQ ID NO:409-SEQ ID NO:419 and sequences fully complementary thereto, which hybridize with HPV MM7; and SEQ ID NO:420-SEQ ID NO:429 and sequences fully complementary thereto, which hybridize with HPV MM8.
The foregoing and other features and advantages of the present invention will be more clearly understood through the following descriptions with reference to the drawings, wherein:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic view showing the detector according to a preferred embodiment of the present invention;
FIG. 2(a) is a schematic view showing the detector according to a preferred embodiment of the present invention;
FIG. 2(b) is a schematic view illustrating the subtype of human papilloma viruses identified by each dot shown in FIG. 2(a);
FIG. 3(a) is the electrophoresis result showing the analyzed PCR products using primer set MY09/MY11 according to a preferred embodiment of the present invention;
FIG. 3(b) is the electrophoresis result showing the analyzed PCR products using primer set MY11/GP6+ according to a preferred embodiment of the present invention;
FIG. 3(c) is the electrophoresis result showing the analyzed PCR products using GAPDH primer set according to a preferred embodiment of the present invention;
FIG. 4(a) is the detecting result on the detector of detecting the PCR products using primer set MY09/MY11 of HPV positive clones according to a preferred embodiment of the present invention;
FIG. 4(b) is detecting result on the detector of detecting the PCR products using primer set MY11/GP6+ of HPV positive clones according to a preferred embodiment of the present invention;
FIG. 5 is a view showing the detecting result on the detectors of detecting samples according to a preferred embodiment of the present invention;
FIG. 6(a) is a schematic view showing the detector according to another preferred embodiment of the present invention;
FIG. 6(b) is a schematic view illustrating the subtype of human papilloma viruses identified by each dot shown in FIG. 6(a);
FIG. 7(a) is a view showing the detector stained with SYBR Green II according to a embodiment of the present invention; and
FIG. 7(b) is a view showing the detecting result on the detectors of detecting samples according to a preferred embodiment of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention will now described more specifically with reference to the following embodiments. Papilloma viruses are small (50-60 nm), nonenveloped, and icosahedral DNA viruses. The DNA of many papilloma viruses, including over 50 human viruses, has been cloned and sequenced. Although there is a high degree of sequence divergence between species, all papilloma viruses share some common features of genome organization. The open reading frames (ORFs) of the virus genomes are designated an early region, a late region, and a long control region (LCR) of transcription. The early region contains genes E1-E8 (not all are present in all species), the late region contains genes L1 and L2 (where “E” denotes early and “L” denotes late), and the long control region (LCR) of transcription includes the promoter and enhancer for the viral early genes and the origin of replication. The early region encodes genes required for viral DNA replication, cellular proliferation, and, in some viruses, cellular transformation. The late region (about 3 kb) codes for the capsid proteins. L1 is the major capsid protein and is relatively well conserved among all the papilloma virus types. The L1 protein is about 500 amino acids in size. L1 probably induces the major humoral and cell-mediated responses to viral infection. The L2 proteins are about 500 amino acids in size, account for only a small proportion of the virion mass, and their function is not yet clear. The LCR region contains an origin of replication with binding sites for E1 and E2 and other cis acting sequences in the promoter and enhancer region.
Generally, PCR has been considered to be the most sensitive method for identifying HPV subtypes in biological samples. A number of different primer combinations amplifying DNA fragment from various regions of the HPV genome have been developed and used for the detection of HPV. However, primers amplifying DNA fragments in the conserved L1 region have become the most widely used in the clinical and epidemiological studies. It is because that certain region of the L1 gene presents a high degree of sequence variability in different HPV subtypes. In other words, the sequence variability among each HPV subtype could be the specific site for identifying each different HPV subtype.
In order to identify the various HPV subtypes, the Applicant focuses on the loci near the end of L1 gene to search the specific sequence variability as mentioned above. More specifically, the PCR fragment synthesized by the primer sets MY11/MY09 (as disclosed in Weimin et al., 1997, J. Clin. Microbiol. 35(6): 1304-1310) in the L1 region is the particular loci ranges where the Applicant refers to find the specific sequence variability for each HPV subtype in the present invention. Since the specific sequence variability for each HPV subtype is not only specific to a particular HPV subtype, but also distinguished from any other HPV subtype, consequently, the probes specifically hybridization with a particular HPV subtype could be selected for identifying or diagnosing HPV subtypes, which is also one of the main purposes of the present invention.
The PCR fragments synthesized by the primer sets MY11/MY09 in the L1 region are about 450 bp in length and had been published. The sequences of the fragments for each HPV subtype described in the invention are publicly available, for example, from the National Center for Biotechnology Information (NCBI) (e.g., www.ncbi.nih.gov). The 39 HPV subtypes identified in the invention includes HPV 6, HPV 11, HPV 16, HPV 18, HPV 26, HPV 31, HPV 32, HPV 33, HPV 35, HPV 37, HPV 39, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 62, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 72, HPV 74, HPV 82, HPV CP8061, HPV CP8034, HPV L1AE5, HPV MM4, HPV MM7 and HPV MM8. The original NCBI Accession number and the loci of the PCR fragments synthesized by the primer sets MY11/MY09 for different HPV subtypes are listed in Table 1:
TABLE 1
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Accession
HPV subtypenumber/length(bp)loci/length(bp)SEQ ID NO.
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HPV 6NC_000904/80126743-7151/409430
HPV 11NC_001525/79316727-7135/409431
HPV 16NC_001526/79046602-7013/412432
HPV 18NC_001357/78576578-6992/415433
HPV 26NC_001583/78556553-6967/415434
HPV 31NC_001527/79126520-6931/412435
HPV 32NC_001586/79616837-7245/409436
HPV 33NC_001528/79096559-6967/409437
HPV 35NC_001529/78516542-6953/412438
HPV 37NC_001687/74216711-7125/415439
HPV 39NC_001535/78336605-7019/415440
HPV 42NC_001534/79176802-7210/409441
HPV 43U12504/45521-435/415442
HPV 44NC_001689/78336647-7061/415443
HPV 45NC_001590/78586582-6996/415444
HPV 51NC_001533/78086486-6897/412445
HPV 52NC_001592/79426623-7031/409446
HPV 53NC_001593/78566614-7022/409447
HPV 54NC_001676/77596561-6972/412448
HPV 55NC_001692/78226647-7061/415449
HPV 56NC_001594/78446559-6967/409450
HPV 58NC_001443/78246608-7016/409451
HPV 59NC_001635/78966571-6985/415452
HPV 61NC_001694/79896732-7146/415453
HPV 62U12499/44921-429/409454
HPV 66NC_001695/78246609-7017/409455
HPV 67D21208/78016584-6992/409456
HPV 68M73258/60422582-2996/415457
HPV 69NC 002171/77006509-6923/415458
HPV 70NC 001711/79056549-6963/415459
HPV 72X94164/79886758-7172/415460
HPV 74U40822/38911613-2027/415461
HPV 82AB027021/78716536-6950/415462
HPV CP8061U12479/45221-432/412463
HPV CP8304U12480/45221-432/412464
HPV L1AE5AF039910/36411-360/350465
HPV MM4U12488/45521-435/415466
HPV MM7U12489/45221-432/412467
HPV MM8U12490/45221-432/412468
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The sequences of the fragments of each HPV subtype described in the invention are listed below:
Human Papilloma Virus subtype 6 (6743-7151/409 bp)
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Human Papilloma Virus subtype 6 (6743-7151/409 bp)
SEQ ID NO 430
tatttgttgg ggtaatcaac tgtttgttac tgtggtagat60
accacacgca gtaccaacat
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gacattatgt gcatccgtaa ctacatcttc cacatacacc120
aattctgatt ataaagagta
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catgcgtcat gtggaagagt atgatttaca atttattttt180
caattatgta gcattacatt
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gtctgctgaa gtaatggcct atattcacac aatgaatccc240
tctgttttgg aagactggaa
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ctttgggtta tcgcctcccc caaatggtac attagaagat300
acctataggt atgtgcagtc
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acaggccatt acctgtcaaa agcccactcc tgaaaaggaa360
aagccagatc cctataagaa
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ccttagtttt tgggaggtta atttaaaaga aaagttttct409
agtgaattg
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Human Papilloma Virus subtype 11 (6727-7135/409
bp)
SEQ ID NO 431
tatttgctgg ggaaaccact tgtttgttac tgtggtagat60
accacacgca gtacaaatat
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gacactatgt gcatctgtgt ctaaatctgc tacatacact120
aattcagatt ataaggaata
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catgcgccat gtggaggagt ttgatttaca gtttattttt180
caattgtgta gcattacatt
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atctgcagaa gtcatggcct atatacacac aatgaatcct240
tctgttttgg aggactggaa
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ctttggttta tcgcctccac caaatggtac actggaggat300
acttatagat atgtacagtc
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acaggccatt acctgtcaga aacccacacc tgaaaaagaa360
aaacaggatc cctataagga
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tatgagtttt tgggaggtta acttaaaaga aaagttttca409
agtgaatta
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Human Papilloma Virus subtype 16 (6602-7013/412
bp)
SEQ ID NO 432
catttgttgg ggtaaccaac tatttgttac tgttgttgat60
actacacgca gtacaaatat
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gtcattatgt gctgccatat ctacttcaga aactacatat120
aaaaatacta actttaagga
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gtacctacga catggggagg aatatgattt acagtttatt180
tttcaactgt gcaaaataac
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cttaactgca gacgttatga catacataca ttctatgaat240
tccactattt tggaggactg
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gaattttggt ctacaacctc ccccaggagg cacactagaa300
gatacttata ggtttgtaac
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ccaggcaatt gcttgtcaaa aacatacacc tccagcacct360
aaagaagatg atccccttaa
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aaaatacact ttttgggaag taaatttaaa ggaaaagttt412
tctgcagacc ta
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Human Papilloma Virus subtype 18 (6587-6992/415
bp)
SEQ ID NO 433
tgtttgctgg cataatcaat tatttgttac tgtggtagat60
accactccca gtaccaattt
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aacaatatgt gcttctacac agtctcctgt acctgggcaa120
tatgatgcta ccaaatttaa
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gcagtatagc agacatgttg aggaatatga tttgcagttt180
atttttcagt tgtgtactat
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tactttaact gcagatgtta tgtcctatat tcatagtatg240
aatagcagta ttttagagga
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ttggaacttt ggtgttcccc cccccccaac tactagtttg300
gtggatacat atcgttttgt
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acaatctgtt gctattacct gtcaaaagga tgctgcaccg360
gctgaaaata aggatcccta
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tgataagtta aagttttgga atgtggattt aaaggaaaag415
ttttctttag actta
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Human Papilloma Virus subtype 26 (6553-6967/415
bp)
SEQ ID NO 434
tatctgttgg ggcaatcaat tgtttgttac ctgtgttgat60
accacccgca gtactaacct
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taccattagt acattatctg cagcatctgc atccactcca120
tttaaaccat ctgattataa
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acaatttata agacatggcg aagaatatga attacaattt180
atatttcagt tgtgtaaaat
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aacacttaca acagatgtta tggcttacat acatttaatg240
aatgcctcca tattggagga
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ttggaatttt ggactaacct tacctcccac tgctagtttg300
gaagatgcct ataggtttat
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taaaaactct gctactacct gtcagcgtaa cgcccctcct360
gtgccaaagg aagatccttt
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tcaaaaattt aaattttggg atgtagattt aaaagaaaaa415
ttttctattg atttg
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Human Papilloma Virus subtype 31 (6520-6931/412
bp)
SEQ ID NO 435
tatttgttgg ggcaatcagt tatttgttac tgtggtagat60
accacacgta gtaccaatat
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gtctgtttgt gctgcaattg caaacagtga tactacattt120
aaaagtagta attttaaaga
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gtatttaaga catggtgagg aatttgattt acaatttata180
tttcagttat gcaaaataac
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attatctgca gacataatga catatattca cagtatgaat240
cctgctattt tggaagattg
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gaattttgga ttgaccacac ctccctcagg ttctttggag300
gatacctata ggtttgtcac
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ctcacaggcc attacatgtc aaaaaactgc cccccaaaag360
cccaaggaag atccatttaa
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agattatgta ttttgggagg ttaatttaaa agaaaagttt412
tctgcagatt ta
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Human Papilloma Virus subtype 32 (6837-7245/409
bp)
SEQ ID NO 436
tatatgttgg ggtaatcaag tgtttctaac tgttgtggat60
actacccgta gtactaacat
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gactgtgtgt gctactgtaa caactgaaga cacatacaag120
tctactaact ttaaggaata
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tctacgccat gcagaggaat atgatataca gtttatattt180
caattgtgca aaattacatt
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atctgtagag gttatgtcat atatccacac catgaatcct240
gacatactag acgattggaa
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tgttggtgta gctccaccgc cctctggtac tttagaagat300
agttatagat ttgtgcagtc
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tcaggccata cgatgtcaag ctaaggtaac agcacctgaa360
aaaaaggatc ctttttctga
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ctattcattt tgggaagtaa atttatctga aaagttttct409
agtgattta
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Human Papilloma Virus subtype 33 (6559-6967/409
bp)
SEQ ID NO 437
tatttgttgg ggcaatcagg tatttgttac tgtggtagat60
accactcgca gtactaatat
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gactttatgc acacaagtaa ctagtgacag tacatataaa120
aatgaaaatt ttaaagaata
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tataagacat gttgaagaat atgatctaca gtttgttttt180
caactatgca aagttacctt
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aactgcagaa gttatgacat atattcatgc tatgaatcca240
gatattttag aagattggca
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atttggttta acacctcctc catctgctag tttacaggat300
acctataggt ttgttacctc
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tcaggctatt acgtgtcaaa aaacagtacc tccaaaggaa360
aaggaagacc ccttaggtaa
|
atatacattt tgggaagtgg atttaaagga aaaattttca409
gcagattta
|
Human Papilloma Virus subtype 35 (6542-6953/412
bp)
SEQ ID NO 438
tatttgttgg agtaaccaat tgtttgttac tgtagttgat60
acaacccgta gtacaaatat
|
gtctgtgtgt tctgctgtgt cttctagtga cagtacatat120
aaaaatgaca attttaagga
|
atatttaagg catggtgaag aatatgattt acagtttatt180
tttcagttat gtaaaataac
|
actaacagca gatgttatga catatattca tagtatgaac240
ccgtccattt tagaggattg
|
gaattttggc cttacaccac cgccttctgg taccttagag300
gacacatatc gctatgtaac
|
atcacaggct gtaacttgtc aaaaacccag tgcaccaaaa360
cctaaagatg atccattaaa
|
aaattatact ttttgggagg ttgatttaaa ggaaaagttt412
tctgcagact ta
|
Human Papilloma Virus subtype 37 (6711-7125/415
bp)
SEQ ID NO 439
cattttatgg ggtaatcaaa tgtttatcac agttgctgat60
aatacacgga acacaaactt
|
ttctattagt gtgtctactg acaatggcga agttacagaa120
tataattctc aaacactcag
|
agaataccta agacatgttg aagaatacca gctttcaatt180
attttacaac tttgtaaagt
|
tcctttaaag gctgaggttt taactcagat aaatgcaatg240
aattctggta tattggaaga
|
gtggcaatta ggatttgtac ctactccaga taattcagta300
catgaccttt ataggtacat
|
taattcaaag gctaccaagt gtcctgatgc agttgttgaa360
aaagaaaagg aagatccctt
|
tgcaaaatat acattttgga atgtagattt aactgaaaaa415
ttatcattgg attta
|
Human Papilloma Virus subtype 39 (6605-7017/415
bp)
SEQ ID NO 440
tatatgttgg cataatcaat tatttcttac tgttgtggac60
actacccgta gtaccaactt
|
tacattatct acctctatag agtcttccat accttctaca120
tatgatcctt ctaagtttaa
|
ggaatatacc aggcacgtgg aggagtatga tttacaattt180
atatttcaac tgtgtactgt
|
cacattaaca actgatgtta tgtcttatat tcacactatg240
aattcctcta tattggacaa
|
ttggaatttt gctgtagctc ctccaccatc tgccagtttg300
gtagacactt acagatacct
|
acagtctgca gccattacat gtcaaaagga tgctccagca360
cctgaaaaga aagatccata
|
tgacggtcta aagttttgga atgttgactt aagggaaaag415
tttagtttgg aactt
|
Human Papilloma Virus subtype 42 (6802-7210/409
bp)
SEQ ID NO 441
tatatgttgg ggaaatcagc tatttttaac tgtggttgat60
actacccgta gtactaacat
|
gactttgtgt gccactgcaa catctggtga tacatataca120
gctgctaatt ttaaggaata
|
tttaagacat gctgaagaat atgatgtgca atttatattt180
caattgtgta aaataacatt
|
aactgttgaa gttatgtcat atatacacaa tatgaatcct240
aacatattag aggagtggaa
|
tgttggtgtt gcaccaccac cttcaggaac tttagaagat300
agttataggt atgtacaatc
|
agaagctatt cgctgtcagg ctaaggtaac aacgccagaa360
aaaaaggatc cttattcaga
|
cttttggttt tgggaggtaa atttatctga aaagttttct409
actgattta
|
Human Papilloma Virus subtype 43 (21-435/415 bp)
SEQ ID NO 442
catttgtttt gggaatcagt tgtttgttac agtggtagat60
accactcgta gtacaaactt
|
gacgttatgt gcctctactg accctactgt gcccagtaca120
tatgacaatg caaagtttaa
|
ggaatacttg cggcatgtgg aagaatatga tctgcagttt180
atatttcaat tatgcataat
|
aacgctaaac ccagaggtta tgacatatat tcatactatg240
gatcccacat tattagagga
|
ctggaatttt ggtgtgtccc cacctgcctc tgcttctttg300
gaagatactt atcgcttttt
|
gtctaacaag gccattgcat gtcaaaaaaa tgctccccca360
aaggaacggg aggatcccta
|
taaaaagtat acattttggg atataaatct tacagaaaag415
ttttctgcac aactt
|
Human Papilloma Virus subtype 44 (6647-7061/415
bp)
SEQ ID NO 443
tatttgttgg ggaaatcagt tatttgttac tgttgtagat60
actacccgta gtacaaacat
|
gacaatatgt gctgccacta cacagtcccc tccgtctaca120
tatactagtg aacaatataa
|
gcaatacatg cgacatgttg aggagtttga cttacaattt180
atgtttcaat tatgtagtat
|
taccttaacg gcggaggtaa tggcctatct tcatactatg240
aatgctggta ttttagaaca
|
gtggaacttt gggttgtcgc cgcccccaaa tggtacctta300
gaggacaaat acagatatgt
|
gcagtcccag gocattacat gtcaaaagcc accccctgaa360
aaggcaaagc aggaccccta
|
tgcaaaatta agtttttggg aggtggatct tagagaaaag415
ttttctagtg agttg
|
Human Papilloma Virus subtype 45 (6582-6996/415
bp)
SEQ ID NO 444
tatttgttgg cataatcagt tgtttgttac tgtagtggac60
actacccgca gtactaattt
|
aacattatgt gcctctacac aaaatcctgt gccaagtaca120
tatgacccta ctaagtttaa
|
gcagtatagt agacatgtgg aggaatatga tttacagttt180
atttttcagt tgtgcactat
|
tactttaact gcagaggtta tgtcatatat ccatagtatg240
aatagtagta tattagaaaa
|
ttggaatttt ggtgtccctc caccacctac tacaagtttg300
gtggatacat atcgttttgt
|
gcaatcagtt gctgttacct gtcaaaagga tactacacct360
ccagaaaagc aggatccata
|
tgataaatta aagttttgga ctgttgacct aaaggaaaaa415
ttttcctccg atttg
|
Human Papilloma Virus subtype 51 (6486-6897/412
bp)
SEQ ID NO 445
catttgctgg aacaatcagc tttttattac ctgtgttgat60
actaccagaa gtacaaattt
|
aactattagc actgccactg ctgcggtttc cccaacattt120
actccaagta actttaagca
|
atatattagg catggggaag agtatgaatt gcaatttatt180
tttcaattat gtaaaattac
|
tttaactaca gaggtaatgg cttatttaca cacaatggat240
cctaccattc ttgaacagtg
|
gaattttgga ttaacattac ctccgtctgc tagtttggag300
gatgcatata ggtttgttag
|
aaatgcagct actagctgtc aaaaggacac ccctccacag360
gctaagccag atcctttggc
|
caaatataaa ttttgggatg ttgatttaaa ggaacgattt412
tctttagatt ta
|
Human Papilloma Virus subtype 52 (6623-7031/409
bp)
SEQ ID NO 446
catatgttgg ggcaatcagt tgtttgtcac agttgtggat60
accactcgta gcactaacat
|
gactttatgt gctgaggtta aaaaggaaag cacatataaa120
aatgaaaatt ttaaggaata
|
ccttcgtcat ggcgaggaat ttgatttaca atttattttt180
caattgtgca aaattacatt
|
aacagctgat gttatgacat acattcataa gatggatgcc240
actattttag aggactggca
|
atttggcctt accccaccac cgtctgcatc tttggaggac300
acatacagat ttgtcacttc
|
tactgctata acttgtcaaa aaaacacacc acctaaagga360
aaggaagatc ctttaaagga
|
ctatatgttt tgggaggtgg atttaaaaga aaagttttct409
gcagattta
|
Human Papilloma Virus subtype 53 (6614-7022/409
bp)
SEQ ID NO 447
catctgttgg aacaatcagt tatttgtaac tgttgtggat60
accaccagga atacaaacat
|
gactctttcc gcaaccacac agtctatgtc tacatataat120
tcaaagcaaa ttaaacagta
|
tgttagacat gcagaggaat atgaattaca atttgtgttt180
caactatgta aaatatccct
|
gtctgctgag gttatggcct atttacatac tatgaattct240
accttactgg aagactggaa
|
tataggtttg tcgcctcctg ttgccactag cttagaggac300
aaatacagat atgtgaaaag
|
tgcagctata acctgtcaaa aggatcagcc ccctcctgaa360
aagcaggacc cactatctaa
|
atataaattt tgggaggtca atttgcaaaa cagtttttct409
gctgatttg
|
Human Papilloma Virus subtype 54 (6561-6972/412
bp)
SEQ ID NO 448
tatttgttgg ggcaatcagg tgtttttaac agttgtagat60
accacccgta gtactaacct
|
aacattgtgt gctacagcat ccacgcagga tagctttaat120
aattctgact ttagggagta
|
tattagacat gtggaggaat atgatttaca gtttatattt180
cagttatgta ccataaccct
|
tacagoagat gttatggcct atattcatgg aatgaatccc240
actattctag aggactggaa
|
ctttggtata acccccccag ctacaagtag tttggaggac300
acatataggt ttgtacagtc
|
acaggccatt gcatgtcaaa agaataatgc ccctgcaaag360
gaaaaggagg atccttacag
|
taaatttaat ttttggactg ttgaccttaa ggaacgattt412
tcatctgacc tt
|
Human Papilloma Virus subtype 55 (6647-7061/415
bp)
SEQ ID NO 449
tatttgttgg gggaatcagt tatttgttac tgttgtagat60
actacacgta gtacaaacat
|
gacaatatgt gctgctacaa ctcagtctcc atctacaaca120
tataatagta cagaatataa
|
acaatacatg cgacatgttg aggagtttga cttacagttt180
atgtttcaat tatgtagtat
|
taccttaact gctgaggtaa tggcctattt acataccatg240
aatcctggta ttttggaaca
|
gtggaacttt gggttgtcgc cacccccaaa tggtacctta300
gaagacaaat acagatatgt
|
gcagtcacag gocattacat gtcaaaagcc tccccctgaa360
aaggcaaagc aggaccccta
|
tgcaaaatta agtttttggg aggtagatct cagagaaaag415
ttttctagtg agtta
|
Human Papilloma Virus subtype 56 (6559-6967/409
bp)
SEQ ID NO 450
catttgctgg ggtaatcaat tatttgttac tgtagtagat60
actactagaa gtactaacat
|
gactattagt actgctacag aacagttaag taaatatgat120
gcacgaaaaa ttaatcagta
|
ccttagacat gtggaggaat atgaattaca atttgttttt180
caattatgca aaattacttt
|
gtctgcagag gttatggcat atttacataa tatgaatgct240
aacctactgg aggactggaa
|
tattgggtta tccccgccag tggccaccag cctagaagat300
aaatatagat atgttagaag
|
cacagctata acatgtcaac gggaacagcc accaacagaa360
aaacaggacc cattagctaa
|
atataaattt tgggatgtta acttacagga cagtttttct419
acagacctg
|
Human Papilloma Virus subtype 58 (6608-7016/409
bp)
SEQ ID NO 451
catttgctgg ggcaatcagt tatttgttac cgtggttgat60
accactcgta gcactaatat
|
gacattatgc actgaagtaa ctaaggaagg tacatataaa120
aatgataatt ttaaggaata
|
tgtacgtcat gttgaagaat atgacttaca gtttgttttt180
cagctttgca aaattacact
|
aactgcagag ataatgacat atatacatac tatggattcc240
aatattttgg aggactggca
|
atttggttta acacctcctc cgtctgccag tttacaggac300
acatatagat ttgttacctc
|
ccaggctatt acttgccaaa aaacagcacc ccctaaagaa360
aaggaagatc cattaaataa
|
atatactttt tgggaggtta acttaaagga aaagttttct409
gcagatcta
|
Human Papilloma Virus subtype 59 (6571-6985/415
bp)
SEQ ID NO 452
tatatgttgg cacaatcaat tgtttttaac agttgtagat60
actactcgca gcaccaatct
|
ttctgtgtgt gcttctacta cttcttctat tcctaatgta120
tacacaccta ccagttttaa
|
agaatatgcc agacatgtgg aggaatttga tttgcagttt180
atatttcaac tgtgtaaaat
|
aacattaact acagaggtaa tgtcatacat tcataatatg240
aataccacta ttttggagga
|
ttggaatttt ggtgttacac cacctcctac tgctagttta300
gttgacacat accgttttgt
|
tcaatctgct gctgtaactt gtcaaaagga caccgcaccg360
ccagttaaac aggaccctta
|
tgacaaacta aagttttggc ctgtagatct taaggaaagg415
ttttctgcag atctt
|
Human Papilloma Virus subtype 61 (6732-7146/415
bp)
SEQ ID NO 453
tatttgttgg tttaatgaat tgtttgtaac cgttgtggat60
accacccgca gtactaattt
|
aaccatttgt actgctacat ccccccctgt atctgaatat120
aaagccacaa gctttaggga
|
atatttgcgc catacagagg agtttgattt gcaatttatt180
tttcagttat gtaaaataca
|
tttaacccct gaaattatgg cctacctaca taatatgaat240
aaggccttgt tggatgactg
|
gaactttggt gtggtaccac caccctctac cagtttagaa300
gacacatata ggtttttgca
|
gtccagagct attacatgtc agaagggtgc tgctgccccg360
ccgcccaagg aggatcgcta
|
tgccaagtta tccttttgga ctgttgattt acgagacaag415
ttttccactg atttg
|
Human Papilloma Virus subtype 62 (21-429/409 bp)
SEQ ID NO 454
tatttgttgg tttaatgaac tgtttgttac tgtggtggat60
actaccagaa gtactaattt
|
tactatttgt accgcctcca ctgctgcagc agaatacacg120
gctaccaact ttagggaatt
|
tttgcgacac acggaggaat ttgatttgca atttatattt180
caattgtgca aaatacagtt
|
aacccccgaa attatggcct acctgcataa tatgaacaag240
gaccttttgg atgactggaa
|
ctttggggtt ttacctcccc cttccactag tttagatgag300
acatatcact atttcgagtc
|
tcgggctatt acatgtcaaa gggggctgcc tacccgtccc360
aaggtggacc cgtatgcgca
|
aatgacattt tggactgtgg atcttaagga caagttgtct409
actgatttg
|
Human Papilloma Virus subtype 66 (6609-7017/409
bp)
SEQ ID NO 455
catatgctgg ggtaatcagg tatttgttac tgttgtggat60
actaccagaa gcaccaacat
|
gactattaat gcagctaaaa gcacattaac taaatatgat120
gcccgtgaaa tcaatcaata
|
ccttcgccat gtggaggaat atgaactaca gtttgtgttt180
caactttgta aaataacctt
|
aactgcagaa gttatggcat atttgcataa tatgaataat240
actttattag acgattggaa
|
tattggctta tccccaccag ttgcaactag cttagaggat300
aaatataggt atattaaaag
|
cacagctatt acatgtcaga gggaacagcc ccctgcagaa360
aagcaggatc ccctggctaa
|
atataagttt tgggaagtta atttacagga cagcttttct409
gcagacctg
|
Human Papilloma Virus subtype 67 (6584-6992/409
bp)
SEQ ID NO 456
tatatgctgg ggtaatcaaa tatttgttac tgttgtagac60
actacacgta gtaccaacat
|
gactttatgt tctgaggaaa aatcagaggc tacatacaaa120
aatgaaaact ttaaggaata
|
ccttagacat gtggaagaat atgatttgca gtttatattt180
cagctgtgca aaatatccct
|
tactgcaaat gttatgcaat acatacacac catgaatcca240
gatatattag aggactggca
|
atttggcctt acaccacctc cttcaggtaa tttacaggac300
acatatagat ttgttacctc
|
gcaggctatt acctgtcaaa aaacatcccc tccaacagca360
aaggaagatc ctcttaaaaa
|
gtacagtttt tgggaaatca atttaaagga aaaattttct409
gcagattta
|
Human Papilloma Virus subtype 68 (2582-2996/415
bp)
SEQ ID NO 457
tatttgttgg cataatcaat tatttcttac tgttgtggat60
accactcgca gtaccaattt
|
tactttgtct actactactg aatcagctgt accaaatatt120
tatgatccta ataaatttaa
|
ggaatatatt aggcatgttg aggaatatga tttgcaattt180
atatttcagt tgtgtactat
|
aacattgtcc actgatgtaa tgtcctatat acatactatg240
aatcctgcta ttttggatga
|
ttggaatttt ggtgttgccc ctccaccatc tgctagtctt300
gtagatacat accgctatct
|
gcaatcagca gcaattacat gtcaaaaaga cgcccctgca360
cctactaaaa aggatccata
|
tgatggctta aacttttgga atgtaaattt aaaggaaaag415
tttagttctg aactg
|
Human Papilloma Virus subtype 69 (6509-6923/415
bp)
SEQ ID NO 458
catttgttgg ggcaaccaat tgtttgttac ttgtgtagat60
actacccgca gtaccaacct
|
cactattagt actgtatctg cacaatctgc atctgccact120
tttaaaccat cagattataa
|
gcagtttata aggcatggtg aggaatatga attacagttt180
atatttcaat tgtgtaaaat
|
tactcttacc actgatgtaa tggcctatat ccatacaatg240
aattctacta ttttggaaaa
|
ttggaatttt ggccttacct tgcctcctac tgctagtttg300
gaagatgcat ataggtttat
|
taaaaattca gctactacat gtcaacgcga tgcccctgca360
cagcccaagg aggatccatt
|
tagtaaatta aaattttggg acgttgatct taaagaaaag415
ttttctattg attta
|
Human Papilloma Virus subtype 70 (6549-6963/415
bp)
SEQ ID NO 459
catttgttgg cataaccagt tgtttattac tgtggtggac60
actacacgta gtactaattt
|
tacattgtct gcctgcaccg aaacggccat acctgctgta120
tatagcccta caaagtttaa
|
ggaatatact aggcatgtgg aggaatatga tttacaattt180
atatttcaat tgtgtactat
|
cacattaact gctgacgtta tggcctacat ccatactatg240
aatcctgcaa ttttggacaa
|
ttggaatata ggagttaccc ctccaccatc tgcaagcttg300
gtggacacgt ataggtattt
|
acaatcagca gctatagcat gtcaaaagga tgctcctaca360
cctgaaaaaa aggatcccta
|
tgacgattta aaattttgga atgttgattt aaaggaaaag415
tttagtacag aacta
|
Human Papilloma Virus subtype 72 (6758-7172/415
bp)
SEQ ID NO 460
catctgttgg tttaatgagc tttttgtgac agttgtagat60
actactcgca gtactaatgt
|
aactatttgt actgccacag cgtcctctgt atcagaatat120
acagcttcta attttcgtga
|
gtatcttcgc cacactgagg aatttgattt gcagtttata180
tttcaactgt gtaaaattca
|
cttaactcct gaaattatgg cctacttgca caatatgaat240
aaggccttat tggatgactg
|
gaattttggt gtggtgcctc ctccttctac cagtttggat300
gatacctata ggtttttgca
|
gtctcgtgcc attacctgtc aaaagggggc tgccacccct360
cctcctaaag aagatccata
|
tgctaactta tccttttgga ctgtggattt aaaggacaaa415
ttttccactg acttg
|
Human Papilloma Virus subtype 74 (1613-2027/415
bp)
SEQ ID NO 461
tatttgttgg ggtaatcaat tatttgttac agttgtggat60
accacacgca gtactaacat
|
gactgtgtgt gctcctacct cacaatcgcc ttctgctaca120
tataatagtt cagactacaa
|
acaatacatg cgacatgtgg aggaatttga tttgcaattt180
atttttcaat tatgtagtat
|
taagttaact gctgaggtta tggcctatat tcatactatg240
aatcctacag ttttagaaga
|
gtggaacttt gggctaacgc ctccccccaa tggtacttta300
gaagacacct acagatatgt
|
gcagtcccag gctattacat gtcaaaaacc tacgcctgat360
aaagcaaagc ccaatcccta
|
tgcaaattta agtttttggg aagttaatct taaggaaaag415
ttttctagtg aatta
|
Human Papilloma Virus subtype 82 (6536-6950/415
bp)
SEQ ID NO 462
catttgctgg aataatcagc tttttattac ttgtgttgac60
actactaaaa gtaccaattt
|
aaccattagc actgctgtta ctccatctgt tgcacaaaca120
tttactccag caaactttaa
|
gcagtacatt aggcatgggg aagaatatga attgcaattt180
atatttcaat tgtgtaaaat
|
cactttaact actgaaatta tggcttacct gcacaccatg240
gattctacaa ttttagaaca
|
gtggaatttt ggattaacat tgcccccctc cgctagtttg300
gaggatgcct atcgatttgt
|
aaaaaatgca gcaacatcct gtcacaagga cagtcctcca360
caggctaaag aagacccttt
|
ggcaaaatat aaattttgga atgtagacct taaggaacgc415
ttttctttgg atttg
|
Human Papilloma Virus subtype CP8061 (21-432/412
bp)
SEQ ID NO 463
catttgttgg ggcaatcagc tttttgtaac agttgtggac60
acatcacgta gtacaaatat
|
gtccatctgt gctaccaaaa ctgttgagtc tacatataaa120
gcctctagtt tcatggaata
|
tttgagacat ggagaagaat ttgatttgca atttatattt180
caactatgtg ttattaattt
|
aacagctgaa attatggcct acttacatcg catggatgct240
acattactgg aggactggaa
|
tttttggttc ttaccacctc ctactgctag tcttggtgat300
acctaccgct ttttacagtc
|
tcaggccata acctgtcaga aaaacagtcc tcctcctgca360
gaaaaaaagg acccctatgc
|
agatcttaca ttttgggagg tggatttaaa ggagcggttt412
tcactagaat tg
|
Human Papilloma Virus subtype CP8304 (21-432/412
bp)
SEQ ID NO 464
tatttgttgg tttaatgaaa tgtttgttac agtggtggat60
actaccagaa gcaccaattt
|
tactatttgc acagctacat ctgctgctgc agaatacaag120
gcctctaact ttaaggaatt
|
tctgcgccat acagaggaat atgatttgca gtttattttc180
caattatgta aaatacagtt
|
aacaccagaa attatggcct acttacataa tatgaacaag240
gcactgttgg atgattggaa
|
ttttggtgtg ttgccacctc cttccaccag tttagatgac300
acatatcgct ttttacagtc
|
tcgggccatt acctgtcaaa agggtgctgc tgcccctgcg360
cccaaagagg acccttatgc
|
cgacatgtca ttttggacag ttgaccttaa ggacaagttg412
tctactgatt tg
|
Human Papilloma Virus subtype L1AE5 (11-360/350
bp)
SEQ ID NO 465
ggcacaacca attatttata actgtggtag acacaacacg60
tagtaccaat cttaccttat
|
ctactgcaac tactaatcca gttccatcta tatatgaacc120
ttctaaattt aaggaataca
|
cacgccatgt agaggaatat gatttacaat ttatatttca180
attgtgtaaa attacactta
|
ctactgatgt tatgtcttat atacataaca tggatcctac240
tattttagat agttggaatt
|
ttggtgttag tcctccccca tctgctagct tagtagatac300
atataggttt ttacagtcat
|
ctgccattac atgtcagaag gatgtggttg ttccacaaaa350
aaaggatcca
|
Human Papilloma Virus subtype MM4 (21-435/415 bp)
SEQ ID NO 466
catttgctgg aataatcagc tttttattac ttgtgttgac60
actactagaa gtaccaattt
|
aaccattagc actgctgtta ctcaatctgt tgcacaaaca120
tttactccag caaactttaa
|
gcaatacatt aggcatgggg aagaatatga attgcaattt180
atatttcaat tgtgtaaaat
|
cactttaact actgaaatta tggcttacct gcacaccatg240
gattctacaa ttttagaaca
|
gtggaatttt ggattaacct tgcccccctc agctagtttg300
gaggatgcct atcgatttgt
|
aaaaaatgca gcaacatcct gtcacaagga cagtcctcca360
caggctaaac aagacccttt
|
ggcaaaatat aaattttgga atgtagacct taaggaacgc415
ttttctttgg atttg
|
Human Papilloma Virus subtype MM7 (21-432/412 bp)
SEQ ID NO 467
catttgttgg tttaatgagt tatttgttac agttgtagat60
actacccgca gtaccaatat
|
tactatttca gctgctgcta cacaggctaa tgaatacaca120
gcctctaact ttaaggaata
|
cctccgccac accgaggaat atgacttaca ggttatattg180
caactttgca aaatacatct
|
tacccctgaa attatggcat acctacatag tatgaatgaa240
catttattgg atgagtggaa
|
ttttggcgtg ttaccacctc cttccaccag ccttgatgat300
acctatcgct atctgcagtc
|
ccgtgctatt acctgccaaa agggtccttc cgcccctgcc360
cctaaaaagg atccttatga
|
tggccttgta ttttgggagg ttgatttaaa ggacaaacta412
tccacagatt tg
|
Human Papilloma Virus subtype MM8 (21-432/412 bp)
SEQ ID NO 468
tatatgctgg tttaatcaat tgtttgtcac ggtggtggat60
accacccgca gcaccaattt
|
tactattagt gctgctacca acaccgaatc agaatataaa120
cctaccaatt ttaaggaata
|
cctaagacat gtggaggaat atgatttgca gtttatattc180
cagttgtgta aggtccgtct
|
gactccagag gtcatgtcct atttacatac tatgaatgac240
tccttattag atgagtggaa
|
ttttggtgtt gtgccccctc cctccacaag tttagatgat300
acctataggt acttgcagtc
|
tcgcgccatt acttgccaaa agggggccgc cgccgccaag360
cctaaggaag atccttatgc
|
tggcatgtcc ttttgggatg tagatttaaa ggacaagttt412
tctactgatt tg
In order to find the specific probes for identifying or diagnosing HPV subtypes, some sequence analysis software are used for finding the variety sites among the above listed sequences of different HPV subtypes, e.g., DNASTAR. The above 450-bp sequences of 39 HPV subtypes are respectively divided into several fragments and analyzed by the software. Preferably, the genetic identify compared to other HPV subtypes must be lower than 30% for finding suitable probes with high specificity. After identifying the variety sites having low genetic identity in sequences of each HPV subtype, the probes for each HPV subtype are respectively designed to specifically hybridize with these variety sites. Then, the designed probes are tested for their specificities to the corresponding HPV subtypes respectively. Preferably, the probes are 15-30 base pairs in length. Ultimately, 9-12 probes with high specificity are found for each HPV subtype. The sequences of the probes for each- HPV subtype are listed below.
|
|
UZ,14/19 HPV 6
SEQ ID
NO5′→3′Locus in HPV 6
|
1CATCCGTAACTACATCTTCC6814-6833
|
2ATCCGTAACTACATCTTCCA6815-6834
|
3CTACATCTTCCACATACACCAA6823-6844
|
4CATCTTCCACATACACCAAT6826-6845
|
5ATCTTCCACATACACCAATT6827-6846
|
6CCACATACACCAATTCTGAT6832-6851
|
7TAGCATTACATTGTCTGCTGAAG6911-6933
|
8TCCCTCTGTTTTGGAAGAC6959-6977
|
09GTTATCGCCTCCCCCAAATGGTACAT6989-7014
|
10CTATAGGTATGTGCAGTCACAG7025-7046
|
11GCCCACTCCTGAAAAGGAA7064-7082
|
12CTATAAGAACCTTAGT7094-7109
|
HPV 11
SEQ ID
NO5′→3′Locus in HPV 11
|
13ATCTGTGTCTAAATC6799-6813
|
14TCTGTGTCTAAATCTGCTAC6800-6819
|
15ATCTGTGTCTAAATCTGCTACATACA6799-6824
|
16TGCATCTGTGTCTAAATCTG6796-6815
|
17AAATCTGCTACATACACTAA6809-6828
|
18CTAAATCTGCTACATACACTA6807-6827
|
19CTACATACACTAATTCAGAT6816-6835
|
20TAGCATTACATTATCTGCAGAAG6895-6917
|
21TCCTTCTGTTTTGGAGGAC6943-6961
|
22TTTATCGCCTCCACCAAATGGTACAC6973-6998
|
23TTATAGATATGTACAGTCACAGGCC7009-7033
|
24ACCCACACCTGAAAAAGAAAAAC7048-7070
|
HPV 16
SEQ ID
NO5′→3′Locus in HPV 16
|
25TATGTCATTATGTGCTGCCA6659-6678
|
26GTGCTGCCATATCTACTTCA6670-6689
|
27TGCCATATCTACTTC6674-6688
|
28TATCTACTTCAGAAACTACA6679-6698
|
29CTACTTCAGAAACTACATATAA6682-6703
|
30ATAAAAATACTAACTTTAAG6700-6719
|
31CAAAATAACCTTAACTGCAGACG6773-6795
|
32TTCCACTATTTTGGAGGAC6821-6839
|
33TCTACAACCTCCCCCAGGAGGCACAC6851-6876
|
34TTATAGGTTTGTAACCCAG6887-6905
|
35ACATACACCTCCAGCACCT6923-6941
|
36CCTTAAAAAATACACT6956-6971
|
HPV 18
SEQ ID
NO5′→3′Locus in HPV 18
|
37TTCTACACAGTCTCC6650-6664
|
38CAGTCTCCTGTACCTGGGCA6657-6676
|
39AGTCTCCTGTACCTGGGCAA6658-6677
|
40TCTCCTGTACCTGGGCAATATGA6660-6682
|
41CTGTACCTGGGCAATATGAT6664-6683
|
42ATGATGCTACCAAATTTAAG6679-6698
|
43TACTATTACTTTAACTGCAGATG6752-6774
|
44TAGCAGTATTTTAGAGGAT6800-6818
|
45TGTTCCCCCCCCCCCAACTACTAGTT6830-6855
|
46ATATCGTTTTGTACAATCTGTT6866-6887
|
47GGATGCTGCACCGGCTGAA6905-6923
|
48CTATGATAAGTTAAAG6935-6950
|
|
HPV 26
SEQ ID
NO5′→3′Locus in HPV 26
|
49TAGTACATTATCTGCAGCAT6619-6638
|
50ATTATCTGCAGCATC6625-6639
|
51TGCAGCATCTGCATCCACTC6631-6650
|
52GCATCTGCATCCACTCCATTTAAA6635-6658
|
53CTCCATTTAAACCATCTGAT6648-6667
|
54TAAAATAACACTTACAACAGATG6727-6749
|
55TGCCTCCATATTGGAGGAT6775-6793
|
56ACTAACCTTACCTCCCACTGCTAGTT6805-6830
|
57CTATAGGTTTATTAAAAACTCT6841-6862
|
58TAACGCCCCTCCTGTGCCA6880-6898
|
HPV 31
SEQ ID
NO5′→3′Locus in HPV 31
|
59TGCAATTGCAAACAG6592-6606
|
60GCAATTGCAAACAGTGATAC6593-6612
|
61CAATTGCAAACAGTGATACT6594-6613
|
62GCAAACAGTGATACTACATTTAA6599-6621
|
63CTACATTTAAAAGTAGTAAT6612-6631
|
64CAAAATAACATTATCTGCAGACA6691-6713
|
65TCCTGCTATTTTGGAAGAT6739-6757
|
66ATTGACCACACCTCCCTCAGGTTCTT6769-6794
|
67CTATAGGTTTGTCACCTCACAG6805-6826
|
68AACTGCCCCCCAAAAGCCC6844-6862
|
HPV 32
SEQ ID
NO5′→3′Locus in HPV 32
|
69TGCTACTGTAACAACTGAAG6906-6925
|
70GCTACTGTAACAACTGAAGA6907-6926
|
71TACTGTAACAACTGA6909-6923
|
72ACTGTAACAACTGAAGACAC6910-6929
|
73CAACTGAAGACACATACAAGTC6917-6938
|
74CAAAATTACATTATCTGTAGAGG7005-7027
|
75TCCTGACATACTAGACGAT7053-7071
|
76TGTAGCTCCACCGCCCTCTGGTACTT7083-7108
|
77TTATAGATTTGTGCAGTCTCAG7119-7140
|
78TAAGGTAACAGCACCTGAA7158-7176
|
79TTTTTCTGACTATTCA7188-7203
|
HPV 33
SEQ ID
NO5′→3′Locus in HPV 33
|
80TATGCACACAAGTAACTAGT6624-6643
|
81CACACAAGTAACTAG6628-6642
|
82ACAAGTAACTAGTGACAGTA6631-6650
|
83GTAACTAGTGACAGTACATATAA6635-6657
|
84GTACATATAAAAATGAAAAT6648-6667
|
85CAAAGTTACCTTAACTGCAGAAG6727-6749
|
86TCCAGATATTTTAGAAGAT6775-6793
|
87TTTAACACCTCCTCCATCTGCTAGTT6805-6830
|
88CTATAGGTTTGTTACCTCTCAG6841-6862
|
89AACAGTACCTCCAAAGGAA6880-6898
|
90CTTAGGTAAATATACA6910-6925
|
HPV 35
SEQ ID
NO5′→3′Locus in HPV 35
|
91TCTGCTGTGTCTTCTAGTGA6612-6631
|
92TGCTGTGTCTTCTAG6614-6628
|
93GTGTCTTCTAGTGACAGTAC6618-6637
|
94CTTCTAGTGACAGTACATATAAA6622-6644
|
95GTACATATAAAAATGACAAT6634-6653
|
96TAAAATAACACTAACAGCAGATG6713-6735
|
97CCCGTCCATTTTAGAGGAT6761-6779
|
98CCTTACACCACCGCCTTCTGGTACCT6791-6816
|
99ATATCGCTATGTAACATCACAG6827-6848
|
100ACCCAGTGCACCAAAACCT6866-6884
|
HPV 37
SEQ ID
NO5′→3′Locus in HPV 37
|
101TGTCTACTGACAATG6782-6796
|
102TGTCTACTGACAATGGCGAA6782-6801
|
103TGACAATGGCGAAGTTACAG6789-6808
|
104GACAATGGCGAAGTTACAGA6790-6809
|
105AATGGCGAAGTTACAGAATA6793-6812
|
106CAGAATATAATTCTCAAACA6806-6825
|
107TAAAGTTCCTTTAAAGGCTGAGG6885-6907
|
108TTCTGGTATATTGGAAGAG6933-6951
|
109ATTTGTACCTACTCCAGATAATTCAG6963-6988
|
110TTATAGGTACATTAATTCAAAG6999-7020
|
111TGCAGTTGTTGAAAAAGAA7038-7056
|
112CTTTGCAAAATATACA7068-7083
|
HPV 39
SEQ ID
NO5′→3′Locus in HPV 39
|
113CTCTATAGAGTCTTC6677-6691
|
114TAGAGTCTTCCATACCTTCT6682-6701
|
115ATAGAGTCTTCCATACCTTC6681-6700
|
116GTCTTCCATACCTTCTACATATG6686-6708
|
117CTACATATGATCCTTCTAAG6700-6719
|
118TACTGTCACATTAACAACTGATG6779-6801
|
119TTCCTCTATATTGGACAA6827-6844
|
120TGTAGCTCCTCCACCATCTGCCAGTT6857-6882
|
121TTACAGATACCTACAGTCTGCA6893-6914
|
122GGATGCTCCAGCACCTGAA6932-6950
|
123ATATGACGGTCTAAAG6962-6977
|
HPV 42
SEQ ID
NO5′→3′Locus in HPV 42
|
124TATATGTTGGGGAAATCAGCTA6802-6823
|
125CACTGCAACATCTGGTGATA6874-6893
|
126GCAACATCTGGTGATACATATACAG6878-6907
CTGCT
|
127CATTAACTGTTGAAGTTATGTCA6978-7000
|
128CCTAACATATTAGAGGAGTGGAATG7019-7044
T
|
129CACCACCACCTTCAGGAACT7053-7072
|
130GTTATAGGTATGTACAATCAGAAG7083-7106
|
131GCTAAGGTAACAACGCCAGAAAAAA7121-7150
AGGAT
|
132CAGACTTTTGGTTTTGGGAGGTAA7158-7181
|
133GAAAAGTTTTCTACTGATTTA7190-7210
|
HPV 43
SEQ ID
NO5′→3′Locus in HPV 43
|
134CATTTGTTTTGGGAATCAGTTG 21-42
|
135TGACCCTACTGTGCCCAGTA 99-118
|
136ACTGTGCCCAGTACATATGACAATGC 106-135
AAAG
|
137GTTTATATTTCAATTATGCATAA 177-199
|
138CCAGAGGTTATGACATATATT 211-231
|
139CCCACATTATTAGAGGACTGGAA 244-266
|
140CCACCTGCCTCTGCTTCTTTG 280-300
|
141CGCTTTTTGTCTAACAAGGCCATTG 313-337
|
142CCAAAGGAACGGGAGGATCCCTA 358-380
|
143CTTACAGAAAAGTTTTCTGCACAAC 409-433
|
HPV 44
SEQ ID
NO5′→3′Locus in HPV 40
|
144TGCCACTACACAGTC6719-6733
|
145CTACACAGTCCCCTCCGTCT6724-6743
|
146TGCCACTACACAGTCCCCTC6719-6738
|
147CAGTCCCCTCCGTCTACATATA6729-6750
|
148CTACATATACTAGTGAACAA6742-6761
|
149TAGTATTACCTTAACGGCGGAGG6821-6843
|
150TGCTGGTATTTTAGAACAG6869-6887
|
151GTTGTCGCCGCCCCCAAATGGTACC6899-6924
T
|
152ATACAGATATGTGCAGTCCCAG6935-6956
|
153GCCACCCCCTGAAAAGGCA6974-6992
|
154CTATGCAAAATTAAGT7004-7019
|
HPV 45
SEQ ID
NO5′→3′Locus in HPV 45
|
155TGCCTCTACACAAAATCCTG6651-6670
|
156CTCTACACAAAATCC6654-6668
|
157ACAAAATCCTGTGCCAAGTA6660-6679
|
158CAAAATCCTGTGCCAAGTAC6661-6680
|
159AATCCTGTGCCAAGTACATATG6664-6685
|
160GTACATATGACCCTACTAAG6677-6696
|
161CACTATTACTTTAACTGCAGAGG6756-6778
|
162TAGTAGTATATTAGAAAAT6804-6822
|
163TGTCCCTCCACCACCTACTACAAGTT6834-6859
|
164ATATCGTTTTGTGCAATCAGTT6870-6891
|
165GGATACTACACCTCCAGAA6909-6927
|
HPV 51
SEQ ID
NO5′→3′Locus in HPV 51
|
166CACTGCCACTGCTGCGGTTT6555-6574
|
167TGCCACTGCTGCGGT6558-6572
|
168CACTGCTGCGGTTTCCCCAA6561-6580
|
169CCACTGCTGCGGTTTCCCCA6560-6579
|
170CTGCGGTTTCCCCAACATTTAC6566-6587
|
171CAACATTTACTCCAAGTAAC6578-6597
|
172TAAAATTACTTTAACTACAGAGG6657-6679
|
173TCCTACCATTCTTGAACAG6705-6723
|
174ATTAACATTACCTCCGTCTGCTAGTT6735-6760
|
175ATATAGGTTTGTTAGAAATGCA6771-6792
|
176GGACACCCCTCCACAGGCT6810-6828
|
177TTTGGCCAAATATAAA6840-6855
|
HPV 52
SEQ ID
NO5′→3′Locus in HPV 52
|
178TGAGGTTAAAAAGGA6695-6709
|
179TGAGGTTAAAAAGGAAAGCA6695-6714
|
180GAGGTTAAAAAGGAAAGCAC6696-6715
|
181TTAAAAAGGAAAGCACATAT6700-6719
|
182AAAGGAAAGCACATATAAAAAT6704-6725
|
183GCACATATAAAAATGAAAAT6712-6731
|
184CAAAATTACATTAACAGCTGATG6791-6813
|
185TGCCACTATTTTAGAGGAC6839-6857
|
186CCTTACCCCACCACCGTCTGCATCTT6869-6894
|
187ATACAGATTTGTCACTTCTACT6905-6926
|
188AAACACACCACCTAAAGGA6944-6962
|
189TTTAAAGGACTATATG6974-6989
|
HPV 53
SEQ ID
NO5′→3′Locus in HPV 53
|
190TCCGCAACCACACAGTCTAT6681-6700
|
191CCGCAACCACACAGT6682-6696
|
192CCGCAACCACACAGTCTATG6682-6701
|
193CACAGTCTATGTCTACATATAA6691-6712
|
194CTACATATAATTCAAAGCAA6703-6722
|
195TAAAATATCCCTGTCTGCTGAGG6782-6804
|
196TTCTACCTTACTGGAAGAC6830-6848
|
197TTTGTCGCCTCCTGTTGCCACTAGCT6860-6885
|
198ATACAGATATGTGAAAAGTGCA6896-6917
|
199GGATCAGCCCCCTCCTGAA6935-6953
|
HPV 54
SEQ ID
NO5′→3′Locus in HPV 54
|
200TACAGCATCCACGCA6633-6647
|
201CAGCATCCACGCAGGATAGC6635-6654
|
202ACGCAGGATAGCTTTAATAA6643-6662
|
203CACGCAGGATAGCTTTAATA6642-6661
|
204ATAGCTTTAATAATTCTGAC6650-6669
|
205TACCATAACCCTTACAGCAGATG6729-6751
|
206TCCCACTATTCTAGAGGAC6777-6795
|
207TATAACCCCCCCAGCTACAAGTAGT6807-6832
T
|
208ATATAGGTTTGTACAGTCACAG6843-6864
|
209GAATAATGCCCCTGCAAAGGAA6882-6903
|
HPV 55
SEQ ID
NO5′→3′Locus in HPV 55
|
210TTTGTTACTGTTGTAGATACTAC6669-6691
|
211ATGACAATATGTGCTGCTAC6705-6724
|
212GACAATATGTGCTGCTACAA6707-6726
|
213TGCTACAACTCAGTCTCCAT6719-6738
|
214CTACAACTCAGTCTCCATCT6721-6740
|
215ACAACTCAGTCTCCATCTAC6723-6742
|
216ATGTTGAGGAGTTTGACTTA6781-6800
|
217TGTTGAGGAGTTTGACTTAC6782-6801
|
218TGAGGAGTTTGACTTACAGT6785-6804
|
HPV 56
SEQ ID
NO5′→3′Locus in HPV 56
|
219CTGCTACAGAACAGT6630-6644
|
220GCTACAGAACAGTTAAGTAA6632-6651
|
221CAGAACAGTTAAGTAAATAT6636-6655
|
222GAACAGTTAAGTAAATATGATGC6638-6660
|
223GTAAATATGATGCACGAAAA6648-6667
|
224CAAAATTACTTTGTCTGCAGAGG6727-6749
|
225TGCTAACCTACTGGAGGAC6775-6793
|
226GTTATCCCCGCCAGTGGCCACCAGCC6805-5830
|
227ATATAGATATGTTAGAAGCACA6841-6862
|
228GGAACAGCCACCAACAGAA6880-6898
|
HPV 58
SEQ ID
NO5′→3′Locus in HPV 58
|
229ATGCACTGAAGTAACTAAGG6674-6693
|
230CACTGAAGTAACTAAGGAAG6677-6696
|
231TGAAGTAACTAAGGA6680-6694
|
232GAAGTAACTAAGGAAGGTAC6681-6700
|
233CTAAGGAAGGTACATATAAAAA6688-6709
|
234ATAAAAATGATAATTTTAAG6703-6722
|
235CAAAATTACACTAACTGCAGAGA6776-6798
|
236TTCCAATATTTTGGAGGAC6824-6842
|
237TTTAACACCTCCTCCGTCTGCCAGTT6854-6879
|
238ATATAGATTTGTTACCTCCCAG6890-6911
|
239AACAGCACCCCCTAAAGAA6929-6947
|
HPV 59
SEQ ID
NO5′→3′Locus in HPV 59
|
240TTCTACTACTTCTTC6643-6657
|
241ACTACTTCTTCTATTCCTAA6647-6666
|
242ACTTCTTCTATTCCTAATGT6650-6669
|
243TCTTCTATTCCTAATGTATACAC6653-6675
|
244ATGTATACACACCTACCAGT6666-6685
|
245TAAAATAACATTAACTACAGAGG6745-6767
|
246TACCACTATTTTGGAGGAT6793-6811
|
247TGTTACACCACCTCCTACTGCTAGTT6823-6848
|
248ATACCGTTTTGTTCAATCTGCT6859-6880
|
249GGACACCGCACCGCCAGTT6898-6916
|
250TTATGACAAACTAAAG6928-6943
|
HPV 61
SEQ ID
NO5′→3′Locus in HPV 61
|
251CTGCTACATCCCCCC6803-6817
|
252ACATCCCCCCCTGTATCTGA6808-6827
|
253CATCCCCCCCTGTATCTGAA6809-6828
|
254CCCCTGTATCTGAATATAAAGC6815-6836
|
255CTGAATATAAAGCCACAAGC6824-6843
|
256TAAAATACATTTAACCCCTGAAA6903-6925
|
257TAAGGCCTTGTTGGATGAC6951-6969
|
258TGTGGTACCACCACCCTCTACCAGTT6981-7006
|
259ATATAGGTTTTTGCAGTCCAGA7017-7038
|
260GGGTGCTGCTGCCCCGCCGCCC7056-7077
|
261CTATGCCAAGTTATCC7089-7104
|
HPV 62
SEQ ID
NO5′→3′Locus in HPV 62
|
262CCGCCTCCACTGCTG 92-106
|
263GCCTCCACTGCTGCAGCAGA 94-113
|
264CTGCTGCAGCAGAATACACG 101-120
|
265GCAGAATACACGGCTACCAA 109-128
|
266CAGAATACACGGCTACCAAC 110-129
|
267CAAAATACAGTTAACCCCCGAAA 189-211
|
268CAAGGACCTTTTGGATGAC 237-255
|
269GGTTTTACCTCCCCCTTCCACTAGTT 267-292
|
270ATATCACTATTTCGAGTCTCGG 303-324
|
271GGGGCTGCCTACCCGTCCC 342-360
|
272GTATGCGCAAATGACA 372-387
|
HPV 66
SEQ ID
NO5′→3′Locus in HPV 66
|
273CAGCTAAAAGCACAT6680-6694
|
274CAGCTAAAAGCACATTAACT6680-6699
|
275CTAAAAGCACATTAACTAAA6683-6702
|
276TTAACTAAATATGATGCCCG6694-6713
|
277CTAAATATGATGCCCGTGAA6698-6717
|
278TAAAATAACCTTAACTGCAGAAG6777-6799
|
279TAATACTTTATTAGACGAT6825-6843
|
280CTTATCCCCACCAGTTGCAACTAGCT6855-6880
|
281ATATAGGTATATTAAAAGCACA6891-6912
|
282GGAACAGCCCCCTGCAGAA6930-6948
|
283CCTGGCTAAATATAAG6960-6975
|
HPV 67
SEQ ID
NO5′→3′Locus in HPV 67
|
284CTGAGGAAAAATCAG6655-6669
|
285GAGGAAAAATCAGAGGCTAC6657-6676
|
286ATCAGAGGCTACATACAAAAATG6665-6687
|
287AGGAAAAATCAGAGGCTACA6658-6677
|
288CTACATACAAAAATGAAAAC6673-6692
|
289CAAAATATCCCTTACTGCAAATG6752-6774
|
290TCCAGATATATTAGAGGAC6800-6818
|
291CCTTACACCACCTCCTTCAGGTAATT6830-6855
|
292ATATAGATTTGTTACCTCGCAG6866-6887
|
293AACATCCCCTCCAACAGCA6905-6923
|
294TCTTAAAAAGTACAGT6935-6950
|
HPV 68
SEQ ID
NO5′→3′Locus in HPV 68
|
295CTACTACTGAATCAG2653-2667
|
296TGAATCAGCTGTACCAAATA2660-2679
|
297GAATCAGCTGTACCAAATAT2661-2680
|
298CAGCTGTACCAAATATTTATGA2665-2686
|
299ATATTTATGATCCTAATAAA2677-2696
|
300TCCTGCTATTTTGGATGAT2804-2822
|
301TACTATAACATTGTCCACTGATG2756-2778
|
302TGTTGCCCCTCCACCATCTGCTAGTC2834-2859
|
303ATACCGCTATCTGCAATCAGCA2870-2891
|
304AGACGCCCCTGCACCTACT2909-2927
|
305ATATGATGGCTTAAAC2939-2954
|
HPV 69
SEQ ID
NO5′→3′Locus in HPV 69
|
306TATTAGTACTGTATCTGCAC6572-6591
|
307CTGTATCTGCACAAT6580-6594
|
308CTGTATCTGCACAATCTGCA6580-6599
|
309TGCACAATCTGCATCTGCCA6587-6606
|
310CAATCTGCATCTGCCACTTTTA6591-6612
|
311CCACTTTTAAACCATCAGAT6604-6623
|
312TAAAATTACTCTTACCACTGATG6683-6705
|
313TTCTACTATTTTGGAAAAT6731-6749
|
314CCTTACCTTGCCTCCTACTGCTAGT6761-6786
T
|
315ATATAGGTTTATTAAAAATTCA6797-6818
|
316CGATGCCCCTGCACAGCCC6836-6854
|
|
HPV 70
SEQ ID
NO5′→3′Locus in HPV 70
|
317TGTCTGCCTGCACCGAAACG6614-6633
|
318CTGCACCGAAACGGC6621-6635
|
319GAAACGGCCATACCTGCTGT6628-6647
|
320CGAAACGGCCATACCTGCTG6627-6646
|
321CGGCCATACCTGCTGTATATAG6632-6653
|
322CTGTATATAGCCCTACAAAG6644-6663
|
323TACTATCACATTAACTGCTGACG6723-6745
|
324TCCTGCAATTTTGGACAAT6771-6789
|
325AGTTACCCCTCCACCATCTGCAAG6801-6826
CT
|
326GTATAGGTATTTACAATCAGCA6837-6858
|
327GGATGCTCCTACACCTGAA6876-6894
|
328CTATGACGATTTAAAA6906-6921
|
HPV 72
SEQ ID
NO5′→3′Locus in HPV 72
|
329ATCTGTTGGTTTAATGAGCT6759-6778
|
330TTTGTGACAGTTGTAGATAC6780-6799
|
331CTGCCACAGCGTCCT6829-6843
|
332ACAGCGTCCTCTGTATCAGA6834-6853
|
333CCACAGCGTCCTCTGTATCA6832-6851
|
334AGCGTCCTCTGTATCAGAATAT6836-6857
|
335CAGAATATACAGCTTCTAAT6850-6869
|
336TAAAATTCACTTAACTCCTGAAA6929-6951
|
337TAAGGCCTTATTGGATGAC6977-6995
|
338TGTGGTGCCTCCTCCTTCTACCAGTT7007-7032
|
339CTATAGGTTTTTGCAGTCTCGT7043-7064
|
340GGGGGCTGCCACCCCTCCTCCT7082-7103
|
341ATATGCTAACTTATCC7115-7130
|
HPV 74
SEQ ID
NO5′→3′Locus in HPV 74
|
342CCTACCTCACAATCG1686-1700
|
343CTCACAATCGCCTTCTGCTA1691-1710
|
344ACCTCACAATCGCCTTCTGC1689-1708
|
345CAATCGCCTTCTGCTACATATA1695-1716
|
346ACAATCGCCTTCTGCTACATAT1694-1715
|
347CTACATATAATAGTTCAGAC1708-1727
|
348TAGTATTAAGTTAACTGCTGAGG1787-1809
|
349TCCTACAGTTTTAGAAGAG1835-1853
|
350GCTAACGCCTCCCCCCAATGGTACTT1865-1890
|
351CTACAGATATGTGCAGTCCCAG1901-1922
|
352ACCTACGCCTGATAAAGCA1940-1958
|
353CTATGCAAATTTAAGT1970-1985
|
HPV 82
SEQ ID
NO5′→3′Locus in HPV 82
|
354TGCTGTTACTCCATC6608-6622
|
355TGCTGTTACTCCATCTGTTG6608-6627
|
356ACTCCATCTGTTGCACAAAC6615-6634
|
357AAACATTTACTCCAGCAAAC6631-6650
|
358TAAAATCACTTTAACTACTGAAA6710-6732
|
359TTCTACAATTTTAGAACAG6758-6776
|
360ATTAACATTGCCCCCCTCCGCTAGTT6788-6813
|
361CTATCGATTTGTAAAAAATGCA6824-6845
|
362GGACAGTCCTCCACAGGCT6863-6881
|
HPV CP8061
SEQ IDLocus in HPV
NO5′→3′CP8061
|
363TCTGTGCTACCAAAACTGTT 86-105
|
364CTACCAAAACTGTTG 92-106
|
365ACCAAAACTGTTGAGTCTAC 94-113
|
366AACTGTTGAGTCTACATATAAA 99-120
|
367GTTGAGTCTACATATAAAGC 103-122
|
368CTACATATAAAGCCTCTAGT 110-129
|
369TGTTATTAATTTAACAGCTGAAA 189-211
|
370TGCTACATTACTGGAGGAC 237-255
|
371GTTCTTACCACCTCCTACTG 267-286
|
372CTACCGCTTTTTACAGTCTCAG 303-324
|
373AAACAGTCCTCCTCCTGCAGAA 342-363
|
374CTATGCAGATCTTACA 375-390
|
HPV CP8034
SEQ IDLocus in HPV
NO5′→3′CP8034
|
375CAGCTACATCTGCTG 92-106
|
376GCTACATCTGCTGCTGCAGA 94-113
|
377ACATCTGCTGCTGCAGAATACA 97-118
|
378TGCTGCAGAATACAAGGCCT 105-124
|
379GCTGCAGAATACAAGGCCTC 106-125
|
380CAGAATACAAGGCCTCTAAC 110-129
|
381TAAAATACAGTTAACACCAGAAA 189-211
|
382CAAGGCACTGTTGGATGAT 237-255
|
383TGTGTTGCCACCTCCTTCCACCAGTT 267-292
|
384ATATCGCTTTTTACAGTCTCGG 303-324
|
385GGGTGCTGCTGCCCCTGCGCCC 342-363
|
386TTATGCCGACATGTCA 375-390
|
HPV L1AE5
SEQ IDLocus in HPV
NO5′→3′L1AE5
|
387ATCTACTGCAACTACTAATC 69-88
|
388CTGCAACTACTAATC 74-88
|
389CTGCAACTACTAATCCAGTT 74-93
|
390ACTACTAATCCAGTTCCATCTA 79-100
|
391CTAATCCAGTTCCATCTATA 83-102
|
392CTATATATGAACCTTCTAAA 98-117
|
393TAAAATTACACTTACTACTGATG 177-199
|
394TCCTACTATTTTAGATAGT 225-243
|
395TGTTAGTCCTCCCCCATCTGCTAGCT 255-280
|
396ATATAGGTTTTTACAGTCATCT 291-312
|
397GGATGTGGTTGTTCCACAA 330-348
|
HPV MM4
SEQ IDLocus in HPV
NO5′→3′MM4
|
398CTGCTGTTACTCAATCTGTT 92-111
|
399TGCTGTTACTCAATC 93-107
|
400GTTACTCAATCTGTTGCACA 97-116
|
401TGCACAAACATTTACTCCAG 111-130
|
402TTACTCAATCTGTTGCACAAAC 98-119
|
403AAACATTTACTCCAGCAAAC 116-135
|
404TAAAATCACTTTAACTACTGAAA 195-217
|
405TTCTACAATTTTAGAACAG 243-261
|
406ATTAACCTTGCCCCCCTCAGCTAGTT 273-298
|
407CTATCGATTTGTAAAAAATGCA 309-330
|
408GGACAGTCCTCCACAGGCT 348-366
|
HPV MM7
SEQ IDLocus in HPV
NO5′→3′MM7
|
409TGCTGCTACACAGGC 93-107
|
410GCTGCTACACAGGCTAATGA 94-113
|
411TGCTACACAGGCTAATGAAT 96-115
|
412CTACACAGGCTAATGAATACAC 98-119
|
413ATGAATACACAGCCTCTAAC 110-129
|
414CAAAATACATCTTACCCCTGAAA 189-211
|
415TGAACATTTATTGGATGAG 237-255
|
416CGTGTTACCACCTCCTTCCACCAGCC 267-292
|
417CTATCGCTATCTGCAGTCCCGT 303-324
|
418GGGTCCTTCCGCCCCTGCCCCT 342-363
|
419TTATGATGGCCTTGTA 375-390
|
HPV MM8
SEQ IDLocus in HPV
NO5′→3′MM8
|
420TGCTACCAACACCGA 93-107
|
421CTACCAACACCGAATCAGAA 95-114
|
422CCAACACCGAATCAGAATATAA 98-119
|
423CAGAATATAAACCTACCAAT 110-129
|
424TAAGGTCCGTCTGACTCCAGAGG 189-211
|
425TGACTCCTTATTAGATGAG 237-255
|
426TGTTGTGCCCCCTCCCTCCACAAGTT 267-292
|
427CTATAGGTACTTGCAGTCTCGC 303-324
|
428GGGGGCCGCCGCCGCCAAGCCT 342-363
|
429TTATGCTGGCATGTCC 375-390
|
The sequences of the probes listed above are either identical or complementary to the corresponding sequences of HPV subtypes so that the probes can hybridize with the sequences of HPV subtypes perfectly.
According to a preferred embodiment of the present invention, a detector for detecting and simultaneously diagnosing 39 subtypes of human papilloma viruses (HPV) contained in a biological sample is provided. Please refer to FIG. 1. The detector 10 is an oligonucleotide biochip. The detector 10 includes a carrier 11 and a plurality of micro-dots 12 immobilized on the carrier 11. The carrier 11 is a nylon membrane. Each micro-dot 12 is used for identifying one particular HPV subtype. There is at least one oligonucleotide sequence contained in each micro-dot 12 that is specific to one particular HPV subtype. The oligonucleotide sequences are the probes selected from the above list for each HPV subtype respectively. For example, the probe on the carrier 11 could contain at least one sequence, which is selected from SEQ ID NO 1 to SEQ ID NO 12 (shown above), for identifying the subtype 6 of human papilloma viruses (HPV 6).
As described in the above, the probes will hybridize specifically with the L1 gene sequence of the corresponding HPV subtype. Preferably, the probes have a length between 15-30 bases. The oligonucleotide sequences contained in each micro-dot 12 serve as a detection probe, which hybridizes specifically with the L1 gene sequence of the particular HPV subtype to form a hybridization complex as a detection indicator. Therefore, each micro-dot 12 identifies a specific HPV subtype via a corresponding oligonucleotide of the specific HPV subtype, and thereby detecting and simultaneously identifying subtypes of human papilloma viruses. The sequences of the oligonucleotides provided by the present invention are specific to the epidemics of human papilloma viruses. The detector 10 is able to simultaneously identify 39 different HPV subtype that are HPV 6, HPV 11, HPV 16, HPV 18, HPV 26, HPV 31, HPV 32, HPV 33, HPV 35, HPV 37, HPV 39, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 62, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 72, HPV 74, HPV 82, HPV CP8061, HPV CP8034, HPV L1AE5, HPV MM4, HPV MM7 and HPV MM8. Furthermore, the detector 10 includes the micro-dot 12 containing a Glutaldehyde-3-phosphodehydrogenase (GAPDH) gene, which is used as an internal control.
EXAMPLE I
The method for immobilizing or mounting the above mentioned probes (oligonucleotides) on the carrier 11 (the nylon membrane) is described as follows.
1.-TTTTTTTTTTTTTTT (SEQ ID NO 469) is added to the 3′ end of the oligonucleotide provided by the present invention by terminal transferase according to the following steps 1.1 to 1.3.
1.1 Mixing the following components:
|
|
10X NEBuffer 45μl
2.5 mM CoCl25μl
oligonucleotide5˜300pmol
10˜300 mM dATP, dCTP, dTTP or dGTP1μl
Terminal Transferase (20 U/μl)0.5˜5μl
(NEW English BioLabs, M0252S)
Add M.Q. H2O to final volume50μl
|
1.2 The components are mixed at 37° C. for 15-60 minutes.
1.3 10 μl of 0.2 M EDTA (pH 8.0) is added to the mixture to stop the reaction.
2. The oligonucleotide having 3′ end labeling is mounted on the carrier 11 according to the following steps 2.1 to 2.3.
2.1 The oligonucleotide having 3′ end labeling is mounted on the carrier 11 by a needle having a 400 μm wide head. The distance between each dot is 1200 μm.
2.2 The carrier 11 having the dot array 12 thereon is exposed to UV light, and the detector 10 is formed.
2.3 The detector 10 is preserved in a drying box.
EXAMPLE II
According to another preferred embodiment of the present invention, the carrier 11 could be a glass plate. The method for immobilizing or mounting the above mentioned probes (oligonucleotides) on the carrier 11 (glass plate) is described as follows.
1. The surface of the carrier 11 is treated according to the following steps 1.1 to 1.8.
1.1 The carrier 11 is cleaned in non-fluorescent and soft cleaner.
1.2 The clean carrier 11 is immersed in 10% NaOH.
1.3 The carrier 11 is oscillated in double-distilled water, 1% HCl solution and methanol in sequence for 2 minutes, and dried in an oven.
1.4 The carrier 11 is immersed in 1% 3-aminopropyltrimethoxysilane (APTMS) in 95% aqueous acetone at room temperature for about 2 minutes.
1.5 The carrier 11 is washed in acetone, and the carrier 11 is dried in the oven at 110° C. for 45 minutes.
1.6 The dried carrier 11 is immersed in 0.2% 1,4-phenylene diisothiocyanate, wherein the solvent is 10% pyridine in dimethyl formamide), at room temperature for 2 hours.
1.7 The carrier 11 is washed in methanol and acetone, and then the carrier 11 is dried.
1.8 The dried carrier 11 is preserved in a vacuum and dry box.
2. The oligonucleotides provided by the present invention are mounted on the carrier 11 (the glass plate) according to the following steps 2.1 to 2.3.
2.1 The oligonucleotide having 3′ end labeling is mounted on the carrier 11 by a needle having a 400 μm wide head. The distance between each dot is 1200 μm.
2.2 The carrier 11 is immersed in 1% NH4OH solution for about 2 minutes, washed in double-distilled water, and then dried at room temperature. Thus, the detector 10 is formed.
2.3 The detector 10 is preserved in a dried box.
According to the above description, a biochip for specifically identifying the subtypes of human papilloma viruses contained in a biological sample is provided. Please refer to FIG. 2(a). The biochip 20 includes a carrier 21 and a plurality of micro-dots 22 immobilized on the carrier 21. The carrier 21 is a nylon membrane. The actual length of the nylon membrane is about 1.44 cm and the actual width of the nylon membrane is about 0.96 cm. The micro-dots 22 are mounted on the carrier 21 according to the foresaid method, wherein the distance between each dot is about 1.2 mm and the diameter of each dot is about 0.4 mm. Each micro-dot 22 contains at least one oligonucleotide (15˜30 mer), and each micro-dot 22 is used for specifically identifying a specific HPV subtype. The sequence of the oligonucleotide is selected from the foresaid list.
The subtype of human papilloma viruses identified by each dot of the micro-dots 22 is illustrated in FIG. 2(b). SC (system control) presents the PCR product amplified from any subtype of human papilloma viruses and biotin-contained primer. NC (negative control) presents the plants DNA fragment irrelevant to HPV. IN (internal control) presents the sequence 5′-gcccagactgtgggtggcag-3′ (SEQ ID NO 470) of the housekeeping gene, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). In sum, the biochip 20 provided in the present invention is able to detect and simultaneously identify 39 different HPV subtypes contained in the biological sample.
According to another preferred embodiment of the present invention, a method for detecting and simultaneously diagnosing 39 subtypes of human papilloma viruses (HPV) contained in a biological sample is provided. The steps are generally described as follows. First, the L1 gene fragment of human papilloma viruses (HPV) contained in the biological sample is amplified by polymerase chain reaction (PCR) using primers labeled with signaling substance. After the amplification product is obtained, it is hybridized with the detector 11 as describe above to form a hybridization complex. Then, the nonhybridized amplification product is removed from the detector 11. Next, the detector 11 is detected for the existence of the hybridization complex through detecting the signaling substance. The micro-dot 12 having the signaling substance shown thereon means a positive result that the biological sample contains the specific HPV subtypes recognized by the corresponding micro-dot 12. Ultimately, the HPV subtypes contained in the biological sample are thereby detected and simultaneously identified.
The method provided by the present invention for detecting and simultaneously identifying 39 subtypes of human papilloma viruses contained in a sample is described as follows.
EXAMPLE III
1. The biological sample obtained from the patient is treated according to the following steps 1.1 to 1.3.
1.1 The cells are centrifuged at 1,500 rpm at 200□ for 5 minutes.
1.2 The cell pellet is washed in 10 mM Tris (pH 8.5) and dissolved in 8 mM NaOH. Then, the solution is transfer to 1.5 mL micro-tube.
1.3 A proper amount of TreTaq (1U/μl) solution is added to the micro-tube. The reaction is carried out at 95□ for 1 hour. The DNA contained in the sample is obtained after centrifugation at 13,500 rpm, 20□ for 5 minutes. The obtained DNA is preserved at −20□.
EXAMPLE IV
2 The L1 gene fragment of human papilloma viruses (HPV) contained in the biological sample is then amplified by polymerase chain reaction (PCR). The polymerase chain reactions are performed according to the following steps.
2.1 Glutaldehyde-3-phosphodehydrogenase (GAPDH) gene is used as the internal control of the polymerase chain reactions so that it could help confirm whether the detecting protocols are precisely followed. The steps are described according to the following steps 2.1.1 to 2.1.3.
2.1.1 Mixing the following components:
|
|
ReagentStockamountFinal concentration
|
|
Sterile H2O2.6
10X Taq Buffer0.51X Taq Buffer
dNTP2.5mM0.4200μM
Template1
GAP241-51) primer10pmol/μl0.20.4pmol/μl
GAP241-32) primer10pmol/μl0.20.4pmol/μl
ProTaq (PROTECH)5U/μl0.10.1U/μl
Total volume (μl)5
|
1)Gap241-5 (SEQ ID NO 471): CCACCAACTGCTTAGCACCCC
|
2)Gap241-3 (SEQ ID NO 472): TGCAGCGTACTCCCCACATCA
|
3) The proper amount of mineral oil is added to prevent the evaporation.
|
2.1.2 The polymerase chain reaction is performed according to the following programs.
|
|
Program 1Program 2Program 3
|
|
94° C., 15 seconds
|
94° C.,57° C.,72° C.,
3 minutes1 minute5 minutes
|
72° C., 30 seconds
|
40 cycles
|
2.1.3 The product of the polymerase chain reaction is analyzed in 2.5% agarose/EtBr (0.5×TBE).
2.2 The DNA contained in the sample is amplified by the polymerase chain reaction according to the following steps.
2.2.1 Mixing the following components:
|
|
ReagentStockAmountFinal concentration
|
|
Sterile H2O4.7-5.7
10X Taq Buffer11× Taq Buffer
dNTP2.5mM0.8200μM
Template1-2
BSA10mg/ml0.10.1μg/μl
Primer1,2)10pmol/μl0.60.6pmol/μl
Primer1,2)10pmol/μl0.60.6pmol/μl
ProTaq (PROTECH)5U/μl0.20.1U/μl
Total volume (μl)10
|
1)MY09/MY11: Weimin et al., 1997, J. Clin. Microbiol. 35(6): 1304-1310
|
2)MY11/GP6+: Weimin et al., 1997, J. Clin. Microbiol. 35(6): 1304-1310
|
3) The proper amount of mineral oil is added to prevent the evaporation.
|
4) The 5' end of the MY09 and GP6+ primers could be labeled with biotin or Cy5 fluorescent substances.
|
2.2.2 The polymerase chain reaction is performed according to the following programs.
|
|
Program 1Program 2Program 3
|
|
94° C., 45 seconds
|
94° C.,45° C.,72° C.,
3 minutes1 minute5 minutes
|
72° C., 1.5 minutes
|
45 cycles
|
2.2.3 The product of the polymerase chain reaction is analyzed in 2.5% agarose/EtBr (0.5×TBE).
According to the above description, the biochip 20 is used for identifying different HPV subtypes. In one embodiment of the invention, the positive clones of human papilloma viruses are used and detected according to the foresaid method. As previously mentioned, the PCR amplification product could be obtained by different primer sets. One is primer set MY09/MY11, the other is primer set MY11/GP6+. Therefore, the positive clones are respectively amplified by PCR using MY11/MY09 primers and MY11/GP6+ primers. The products of the polymerase chain reaction are analyzed in 2.5% agarose/EtBr, and the electrophoresis results are shown in FIG. 3(a)-(c). FIG. 3(a) shows the electrophoresis result of the analyzed PCR products using primer set MY09/MY11. In FIG. 3(a), M presents DNA marker. Lane 1˜20 present HPV 6, HPV 11, HPV 16, HPV 18, HPV 26, HPV 31, HPV 33, HPV 35, HPV 44, HPV 45, HPV 52, HPV 53, HPV 54, HPV 56, HPV 59, HPV 61, HPV 66, HPV 70, HPV CP8061, and HPV L1AE5 in sequence. FIG. 3(b) shows the electrophoresis result of the analyzed PCR products using primer set MY11/GP6+. In FIG. 3(b), M presents DNA marker. Lane 1˜39 present HPV 6, 11, 16,18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7, and MM8 in sequence. FIG. 3(c) shows the electrophoresis result of the PCR products using GAPDH primer set. Clearly, the electrophoresis results show the PCR products with correct sizes. That is, PCR products using primer set MY09/MY11 is about 450 bp, the PCR products using primer set MY11/GP6+ is about 190 bp, and the PCR products using GAPDH primer set is about 190 bp.
EXAMPLE V
3. When the carrier 11 is a nylon membrane, the detector 10 provided by the present invention is used for identifying the subtypes of human papilloma viruses according to the following hybridization steps.
3.1 The detector 10 is immersed in 2×SSC solution for 5 minutes.
3.2 The detector 10 is immersed in a buffer containing salmon sperm DNA (50 μg/μl), and the oligonucleotides mounted on the detector 10 are pre-hybridized with the salmon sperm DNA at 35□ for 30 minutes.
3.3 The PCR product having biotin labeled thereon is added into and mixed with a buffer containing salmon sperm DNA (50 μg/μl) at 95□ for about 5 minutes. The denatured DNA is placed on ice.
3.4 The denature DNA is added to the detector 10 and hybridized with the oligonucleotides at 35□ for 4 hours or overnight.
3.5 The detector 10 is washed in 2×SSC/1% SDS solution at 35□ for 15 minutes.
3.6 The detector 10 is washed in 0.2×SSC/0.1% SDS solution at 35□ for 15 minutes.
3.7 The detector 10 is treated in 0.5% isolation reagent for 1 hour.
3.8 The detector 10 is treated with avidin-alkalinephosphatase for about 1 hour.
3.9 The detector 10 is washed in 1×PBST solution.
3.10 The detector 10 is washed in Tris/NaCl solution.
3.11 The detector 10 is treated with NBT/BCIP at room temperature to show the reacting dot in blue.
3.12 The blue dot having the specific oligonucleotide sequence presents the specific subtype of human papilloma viruses contained in the sample.
Preferably, the foresaid PCR amplified products shown in FIGS. 3(a)and 3(b) are then respectively detected by the biochip 20 according to the above steps and the results are shown in FIGS. 4(a) and 4(b). FIG. 4(a) shows the detecting result of detecting the PCR products using primer set MY09/MY11 of HPV positive clones. FIG. 4(b) shows the detecting result of detecting the PCR products using primer set MY11/GP6+ of HPV positive clones. When comparing the results shown in FIG. 4(a) and FIG. 3(b) based on the “SC” dot, it is very clear that the biochip 20 can precisely identify the subtype of human papilloma viruses. Take the result of HPV 6 as example. Since this biochip is hybridized with the PCR product amplified from HPV 6 positive clone, there should be 6 positive micro-dots shown on the biochip 20, including 2 SC micro-dots at the corners, 2 SC micro-dots in the central, and 2 micro-dots of HPV 6. The result clearly shows the exact 6 positive micro-dots without any other false positive micro-dot. Obviously, all the results of other biochips in FIGS. 4(a) and 4(b) show a clear and clean result as well. In other words, there is no cross reaction occurred in the detection, which proves that the biochip provided in the present invention has a very high specificity.
In addition, in another embodiment of the invention, the biological sample obtained from the patient is used and detected. The biochip 20 and the detection method described in the above are used for detecting and identifying the HPV subtypes contained in the sample according to the foresaid method. The results are shown in FIG. 5. When comparing the results shown in FIG. 5 and FIG. 3(b) based on the “SC” dot, the results show that HPV 53 is contained in the sample (1), HPV 45 is contained in the sample (2), HPV 52 is contained in the sample (3), and HPV 39 is contained in the sample (4). Therefore, when detecting the biological sample obtained from a patient, it is very clear that the biochip 20 can precisely identify the subtype of human papilloma viruses.
EXAMPLE VI
According to another embodiment of the present invention, the carrier 11 could be a glass plate. When the carrier 11 is a glass plate, the detector 10 provided by the present invention is used for identifying the subtypes of human papilloma viruses according to the following hybridization steps.
4.1 The PCR product having CyS labeled thereon is purified by PCR Clean Up-M System (Viogene, USA), and the PCR product is precipitated in ethanol. Then, the PCR product is dried.
4.2 The precipitated DNA is dissolved in 12 μl of the buffer (2×SSC/0.1% SDS), and centrifugated for 1 minute, and then placed on boiled water for 2 minutes. Then, the mixture is placed on ice for 5 minutes.
4.3 The mixture is centrifugated for 30 seconds, and 10 μl of the mixture is added to the left side of the dot array 22. A cover slice is carefully covered on the dot array from the left side of the dot array to prevent the bubble formation. Then, the detector 10 is place in Humid Chamber (Sigma, USA), and the dot array is faces downward at 35□ for 4 hours or overnight.
4.4 The detector 10 is vertically placed in the solution A (2×SSC/1% SDS), and the detector is slightly oscillated apart from the cover slice. Then, the detector 20 is washed in a shaker at 160 rpm for 12 minutes.
4.5 The detector 10 is washed in the solution B (0.2×SSC/0.1% SDS) and oscillated at 35□ for 12 minutes. The detector 10 is washed in water. Then the detector 10 is dried.
4.6 The dried detector 10 is scanned by GenePix™4000 (Axon, USA), excited by the light having 635 nm of wavelength, and analyzed by GenePixPro 3.0 (Axon, USA).
According to the above description, a biochip for specifically identifying the subtypes of human papilloma viruses contained in a biological sample is provided. Please refer to FIGS. 6(a) and (b). The biochip 30 includes a carrier 31 and a plurality of micro-dots 32 immobilized on the carrier 31. The carrier 31 is a glass plate. The micro-dots 32 are immobilized on the glass plate 31 according to the foresaid method. Each micro-dot 32 contains at least one oligonucleotide (15˜30mer), and each micro-dot 32 is used for specifically identifying a specific HPV subtype. The sequence of the oligonucleotide is selected from the foresaid list. The subtype of human papilloma viruses identified by each dot of the micro-dots 32 is illustrated in FIG. 6(b).
The biochip 30 is stained with SYBR Green II, scanned by GenePix™ 4000 (Axon, USA) and excited by the light having 635 nm of wavelength. The result is shown in FIG. 7(a). Preferably, the foresaid PCR amplified products are then detected by the biochip 30 according to the above steps and the results are shown in FIGS. 7(b). When comparing the results shown in FIG. 7(a) and FIG. 6(b), it is very clear that the biochip 30 can precisely identify the subtype of human papilloma viruses. The result clearly shows the exact positive micro-dots without any other false positive micro-dot. Besides, there is no cross reaction occurred in the detection, which proves that the biochip provided in the present invention has a very high specificity. Therefore, the biochip having different carriers (made of nylon membrane or glass plate) can obtain the same results and same specificities.
According to the above, the drawbacks in the conventional HPV detecting kit do not exist in the HPV detecting kit provided in the present invention. The HPV detecting kit of the present invention is able to diagnose multiple HPV subtypes (up to 39 different subtypes) at the same time, allowing the rapid and reliable detection and identification of HPV possibly present in a biological sample. Besides, an internal control is included in the detector to show whether the detecting process is well handled so that the detecting result is dependable. In addition, HPV detecting kit of the present invention has a high specificity and accuracy. Hence, the present invention not only has a novelty and a progressive nature, but also has an industry utility.
While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.