CN101871014A - HPV (human papillomavirus) detection and typing method - Google Patents
HPV (human papillomavirus) detection and typing method Download PDFInfo
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- CN101871014A CN101871014A CN200910156982A CN200910156982A CN101871014A CN 101871014 A CN101871014 A CN 101871014A CN 200910156982 A CN200910156982 A CN 200910156982A CN 200910156982 A CN200910156982 A CN 200910156982A CN 101871014 A CN101871014 A CN 101871014A
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Abstract
The invention relates to an HPV detection and typing method, which comprises the following steps: carrying out lysis and separation on HPV in a biological sample by using HPV lysate to extract nucleic acid; amplifying an HPVL1 nucleic acid fragment from the nucleic acid obtained in the previous step by using specific primers SEQ ID No.1-SEQ ID No.4; carrying out pyrophosphoric acid sequencing reaction on the obtained HPVL1 nucleic acid fragment, wherein a pyrophosphoric acid sequencing primer is selected from any one or several of primers SEQ ID No.5-SEQ ID No.31; and comparing sequences read by pyrophosphoric acid sequencing with HPV type sequences to judge an HPV typing result. The invention detects and types the HPV in the biological sample by utilizing a DNA (deoxyribonucleic acid) bar code technique and a pyrophosphoric acid sequencing technique and can be used for estimating the carcinogenesis/progression of genital tract cancer and relevant diseases and carries out prognosis guidance on the genital tract cancer and the relevant diseases. The invention has simple and convenient operation, high flux, good specificity, high sensitivity, higher accuracy, high detection speed and low detection cost, and thereby, the invention has wide application prospect.
Description
Technical field
The invention belongs to life science and biological technical field, particularly a kind of by the HPV in DNA barcode technology and the tetra-sodium sequencing technologies detection of biological sample, and it is carried out somatotype.
Background technology
Human papillomavirus is a kind of small-sized dna virus, and genomic dna is a ring-type, the about 8000bp of size.Difference according to its genome structure is divided into more than 100 kind of hypotype at present
[1.2].Zur Hausen in 1974 proposes the HPV infection has substantial connection with cervical cancer, and nineteen ninety-five WHO and IARC are defined as HPV the cause of disease of cervical cancer
[3]Mechanism of causing a disease and the carcinogenic risk of different subtype HPV have nothing in common with each other, and according to the classification of HPV epidemiology, wherein 15 hypotype HPV are high-risk-type, and 3 hypotypes are possible high-risk-type, and 12 hypotypes are low risk
[4,5]Low dangerous type HPV comprises HPV6,11,42,43,44 etc., often cause that benign lesion such as genitalia condyloma comprises low pathology (CIN I) in the epithelium of cervix uteri, high-risk type HPV comprises HPV16,18,31,33,35,39,45,51,52,56,58,59,68, hypotypes such as CP8304, relevant with the generation of cervical cancer and epithelium of cervix uteri inner height pathology (CIN II/III), especially HPV16 and 18 types.HPV infects be the main diseases that takes place of cervical cancer because of, the infection that continues high-risk HPV is the necessary factor of precancerous lesion and the development of wetting property cervical cancer.Because each hypotype HPV virulence and metainfective prognosis have very big-difference, therefore be necessary to set up a kind of easy, quick, sensitive, special genotyping detection method clinically.
Detection to HPV is mainly undertaken by molecular biology method.(hybrid capture is at present uniquely to be applied to clinical HPV detection method through drugs approved by FDA HC-II), but can only distinguishes high-risk and low danger hybrid capture, concrete somatotype, and needing its necessary instrument when using---gene signal enlarges instrument, costs an arm and a leg, and detects the cost height.The water conservancy diversion of clinical present use hybridization rule is because technical defective causes false negative and false positive higher, the fluorescent real time PCR technology is carried out somatotype to HPV and is detected and can not accurately distinguish the HPV hypotype in addition, and may have false positive, flux is low, can not detect a plurality of hypotypes simultaneously; SzuhaiK etc.
[6]The method of SybrGreen and molecular beacon quantitative PCR of using is carried out the somatotype detection to HPV, specificity is fine, and can carry out relative quantification to the HPV of sample, but they only can distinguish 7 HPV hypotypes (HPV-06 ,-11,-16,-18 ,-31 ,-33,-45), can not meet clinical needs far away.
Therefore develop the accurate rapid detection of a kind of energy and also can carry out the method for gene type to HPV, for the examination of reproductive tract tumour, seeming of prevention cervical cancer is particularly necessary.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, provide a kind of quick, accurately, high-throughout HPV detects and classifying method.
A kind of HPV detects and classifying method, it is characterized in that may further comprise the steps:
(a) with the HPV lysate HPV in the biological sample is carried out cracking, separation and Extraction nucleic acid;
(b) with the following Auele Specific Primer HPVL1 nucleic acid fragment that from step (a) gained nucleic acid, increases:
G1：5’-CARGGACATAAYAATGGYATTTGCTG-3’(SEQ?ID?No.1)
G2：5’-CARGGACATAAYAATGGYATTTGTTG-3’(SEQ?ID?No.2)
G3：5’-GAAAAAYAAACTGYAAATCATAYTCCTC-3’(SEQ?ID?No.3)
G4：5’-GAAAAAYAAACTGYAAATCATAYTCTTC-3’(SEQ?ID?No.4)
Wherein G3/G4 is the vitamin H labeled primer;
(c) the HPVL1 nucleic acid fragment that step (b) is obtained carries out the tetra-sodium sequencing reaction, the tetra-sodium sequencing primer be selected from following any one or a few:
HPV6s：5’-AACTACATCTTCCACATACAC-3’(SEQ?ID?No.5)
HPV11s：5’-GTCTAAATCTGCTACATACAC-3’(SEQ?ID?No.6)
HPV16s：5’-ATTATGTGCTGCCATATCTACTT-3’(SEQ?ID?No.7)
HPV18s：5’-ACAGTCTCCTGTACCTGGG-3’(SEQ?ID?No.8)
HPV31s：5’-CCAATATGTCTGTTTGTGCTGC-3’(SEQ?ID?No.9)
HPV33s：5’-GTACTAATATGACTTTATGC-3’(SEQ?ID?No.10)
HPV35s：5’-GTGTTCTGCTGTGTCTTCTAGT-3’(SEQ?ID?No.11)
HPV39s：5’-TATAGAGTCTTCCATACCT-3’(SEQ?ID?No.12)
HPV40s：5’-CCCCACACCAACCCCATATAA-3’(SEQ?ID?No.13)
HPV42s：5’-CATGACTTTGTGTGCCACTG-3’(SEQ?ID?No.14)
HPV43s：5’-CGTTATGTGCCTCTACTGACC-3’(SEQ?ID?No.15)
HPV44s：5’-CTACACAGTCCCCTCC-3’(SEQ?ID?No.16)
HPV45s：5’-CTCTACACAAAATCCTGTG-3’(SEQ?ID?No.17)
HPV51s：5’-CTGCTGCGGTTTCC-3’(SEQ?ID?No.18)
HPV52s：5’-TTTATGTGCTGAGGTTAAAAAG-3’(SEQ?ID?No.19)
HPV53s：5’-CGCAACCACACAGTCTATG-3’(SEQ?ID?No.20)
HPV54s：5’-ACAGCATCCACGCAGG-3’(SEQ?ID?No.21)
HPV56s：5’-CAGAACAGTTAAGTAAATATGAT-3’(SEQ?ID?No.22)
HPV58s：5’-CACTGAAGTAACTAAGG-3’(SEQ?ID?No.23)
HPV59s：5’-TTCTACTACTTCTTCTATTCCTAA-3’(SEQ?ID?No.24)
HPV66s：5’-TAATGCAGCTAAAAGC-3’(SEQ?ID?No.25)
HPV68as：5’-TTTGTCTACTACTACTGAATCA-3’(SEQ?ID?No.26)
HPV70s：5’-AACGGCCATACCTGCTGTATAT-3’(SEQ?ID?No.27)
HPV73s：5’-TAGGTACACAGGCTAGTAG-3’(SEQ?ID?No.28)
HPV83s：5’-GCTACACAGGCTAATGAATACACA-3’(SEQ?ID?No.29)
HPVmm4s：5’-CACTGCTGTTACTCAATCTG-3’(SEQ?ID?No.30)
HPVCP8304s：5’-GCTACATCTGCTGCT-3’(SEQ?ID?No.31)
(d) sequence and the HPV type sequence that the tetra-sodium order-checking is read compared, and judges HPV somatotype result.
Further, the HPV lysate composition described in the step (a) is: 20mmol/L Tris.HCL pH8.0,0.5%Triton (Triton)-X-100,10%chelex (chelate resin)-100.
The described HPV type of step (d) sequence comprises:
HPV6：5’-CAATTCTGATTATAAAGAGTACATGCG-3’(SEQ?ID?No.32)
HPV?11：5’-TAATTCAGATTATAAGGAATA-3’(SEQ?ID?No.33)
HPV16：5’-CAGAAACTACATATAAAAATACTAACTT-3’(SEQ?ID?No.34)
HPV?18：5’-CCTATGATGCTACCAAATTTAA-3’(SEQ?ID?No.35)
HPV31：5’-AATTGCAAACAGTGATACTACATTT-3’(SEQ?ID?No.36)
HPV33：5’-ACACAAGTAACTAGTG-3’(SEQ?ID?No.37)
HPV35：5’-GACAGTACATATAAAAA-3’(SEQ?ID?No.38)
HPV39：5’-TCTACATATGATCCTT-3’(SEQ?ID?No.39)
HPV40：5’-TAACAGTAATTTCAAGGAATATTTGCGT-3’(SEQ?ID?No.40)
HPV42：5’-CAACATCTGGTGATACATATACA-3’(SEQ?ID?No.41)
HPV43：5’-CTACTGTGCCCAGTACATATGACAA-3’(SEQ?ID?No.42)
HPV44：5’-GTCTACATATACTAGTGAACAATATAA-3’(SEQ?ID?No.43)
HPV45：5’-CCAAGTACATATGACCCTA-3’(SEQ?ID?No.44)
HPV51：CCAACATTTACTCCAAGTAACTTTAAGCAAT-3’(SEQ?ID?No.45)
HPV52：5’-GAAAGCACATATAAAAATGAAAATTTTAA-3’(SEQ?ID?No.46)
HPV53：5’-CAGTCTATGTCTAC-3’(SEQ?ID?No.47)
HPV54：5’-ATAGCTATATCTG-3’(SEQ?ID?No.48)
HPV56：5’-GCACGAAAAATTAATCAGTACCTT-3’(SEQ?ID?No.49)
HPV58：5’-AAGGTACATATAAAAATGATAATTTTAAGG-3’(SEQ?ID?No.50)
HPV59：5’-TGTATACACACCTACCAGTTTTAAA-3’(SEQ?ID?No.51)
HPV66：5’-ACATTAACTAAATATGATGCCC-3’(SEQ?ID?No.52)
HPV68a：5’-GCTGTACCAAATATTTATGATCCTAATAAATTT -3’(SEQ IDNo.53)
HPV70：5’-AGCCCTACAAAGTTTAAGGAATATACTAG-3’(SEQ?ID?No.54)
HPV73：5’-CTCTACTACAACGTATGCCAACTCTAA-3’(SEQ?ID?No.55)
HPVmm4：5’-TGCACAAACATTTACTCCAGCAAA-3’(SEQ?ID?No.56)
HPV83：5’-GCCTCTAACTTTAAGGAATACCTCCGC-3’(SEQ?ID?No.57)
HPVCP8304：5’-GCAGAATACAAGGCCTCTAACTTTAA-3’(SEQ?ID?No.58)。
The present invention utilizes DNA barcode technology and tetra-sodium sequencing technologies, to the HPV in the biological specimen detect, somatotype, can be used for assessing the generation development of genital cancers and relative disease and it carried out prognosis instruct.Of the present invention easy and simple to handle, flux is high, specificity good, highly sensitive, accuracy is higher, detection speed is fast, it is low to detect cost, have broad application prospects.
Description of drawings
The tetra-sodium sequencer map that Fig. 1 is to use the inventive method that a testing sample is detected
The tetra-sodium sequencer map that Fig. 2 is to use the inventive method that another testing sample is detected
The tetra-sodium sequencer map that Fig. 3 is to use the inventive method that the 3rd testing sample detected
The tetra-sodium sequencer map that Fig. 4 is to use the inventive method that the 4th testing sample detected
Embodiment
Embodiment 1
The amplification of HPV gene fragment
Get cervical secretions and preserve liquid 1ml, centrifugal 10 minutes of 13000rpm, abandon behind the supernatant with HPV lysate (20mmol/L Tris.HCL pH8.0,0.5%Triton-X-100,10%chelex-100) 20 μ l suspended sediments, 100 ℃ were boiled 10 minutes, 13000rpm gets 2 μ l as HPV gene amplification template after centrifugal 10 minutes, amplification system is as follows: primer G1～G4 (SEQ ID No.1～SEQ ID No.4) gets 3 μ l, 10mmol/L dNTPs 4 μ l, 25mmol/L MgCl respectively
216 μ l, 10 * PCR damping fluid, 2 μ l, cumulative volume 200 μ l divide 4 pipe amplifications; Amplification program is as follows: 94 ℃ of pre-sex change 2min, 40 circulations (94 ℃ of 20s, 50 ℃ of 30s, 72 ℃ of 1min).
Embodiment 2
The preparation of single-stranded template
With aforementioned amplification gained PCR product, every sample was at room temperature hatched 10 minutes according to the magnetic bead that 50 μ l add 3 μ l Streptavidin bag quilts.With PCR product after magnetic bead combines respectively at 75% ethanol, respectively hatched 10 seconds in the NaOH solution of 0.2mol/L and the Tris elutriant, fully reaction back sex change obtains single stranded DNA.To discharge with the strand after magnetic bead combines and be suspended in (100mM Tris-acetate pH7.75,20mM Mg-acetate contain 10pmol mixing sequencing primer SEQ ID No.5～SEQ ID No.31) in the 45 μ l annealing buffers.Under 80 ℃, hatched 2 minutes, at room temperature placed then 5 minutes.
Embodiment 3
The tetra-sodium sequencing reaction
Sequencing reaction is detecting under the SQA pattern of PYROMARK ID (Qiagen company) instrument under 28 ℃ automatically, primer strand is along with the different dNTP (adding of addition sequence: C → T → G → A → C → T → G → A → C → T → G → A) and extending, carrying out along with the reaction of enzymatic, ccd video camera detects the optical signal that sends, and the order-checking collection of illustrative plates as shown in Figure 1.The sequence that records is: CAGAAACTAC, overlap with SEQ IDNo.34, and be judged as the HPV-16 type.
Embodiment 4
The amplification of HPV gene fragment, the preparation of single-stranded template, tetra-sodium sequencing reaction detect another testing sample with embodiment 1～3.The tetra-sodium sequencer map as shown in Figure 2, the sequence that records is: CCGAAACGAAAACTGACT, mononucleotide interpolation order C → T → G → A and SEQ ID No.32-58 are judged as during according to sequencing reaction: HPV-16/HPV-35 polyinfection.
The amplification of HPV gene fragment, the preparation of single-stranded template, tetra-sodium sequencing reaction detect the 3rd testing sample with embodiment 1～3.The tetra-sodium sequencer map as shown in Figure 3, the sequence that records is: GGAACCTGATGACTAAA, mononucleotide interpolation order C → T → G → A and SEQ ID No.32-58 are judged as HPV-35/HPV-68 polyinfection during according to sequencing reaction.
Embodiment 6
The amplification of HPV gene fragment, the preparation of single-stranded template, tetra-sodium sequencing reaction detect the 4th testing sample with embodiment 1～3.The tetra-sodium sequencer map as shown in Figure 4, the sequence that records is: TTATCTGAACTGACT, mononucleotide interpolation order C → T → G → A and SEQ ID No.32-58 are judged as HPV-66/HPV-53 polyinfection during according to sequencing reaction.
Sequence table
＜110〉Hangzhou Ai Dikang medical test center company limited
＜120〉a kind of HPV detects and classifying method
<130>
<160>58
<170>PatentIn?version?3.3
<210>1
<211>26
<212>DNA
＜213〉artificial sequence
<400>1
carggacata?ayaatggyat?ttgctg 26
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<400>2
carggacata?ayaatggyat?ttgttg 26
<210>3
<211>28
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<400>3
gaaaaayaaa?ctgyaaatca?taytcctc 28
<210>4
<211>28
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<400>4
gaaaaayaaa?ctgyaaatca?taytcttc 28
<210>5
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<400>5
aactacatct?tccacataca?c 21
<210>6
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gtctaaatct?gctacataca?c 21
<210>7
<211>23
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<400>7
attatgtgct?gccatatcta?ctt 23
<210>8
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acagtctcct?gtacctggg 19
<210>9
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ccaatatgtc?tgtttgtgct?gc 22
<210>10
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gtactaatat?gactttatgc 20
<210>11
<211>22
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gtgttctgct?gtgtcttcta?gt 22
<210>12
<211>19
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tatagagtct?tccatacct 19
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ccccacacca?accccatata?a 21
<210>14
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catgactttg?tgtgccactg 20
<210>15
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cgttatgtgc?ctctactgac?c 21
<210>16
<211>16
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<400>16
ctacacagtc?ccctcc 16
<210>17
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<400>17
ctctacacaa?aatcctgtg 19
<210>18
<211>14
<212>DNA
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<400>18
ctgctgcggt?ttcc 14
<210>19
<211>22
<212>DNA
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<400>19
tttatgtgct?gaggttaaaa?ag 22
<210>20
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<212>DNA
＜213〉artificial sequence
<400>20
cgcaaccaca?cagtctatg 19
<210>21
<211>16
<212>DNA
＜213〉artificial sequence
<400>21
acagcatcca?cgcagg 16
<210>22
<211>23
<212>DNA
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cagaacagtt?aagtaaatat?gat 23
<210>23
<211>17
<212>DNA
＜213〉artificial sequence
<400>23
cactgaagta?actaagg 17
<210>24
<211>24
<212>DNA
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<400>24
ttctactact?tcttctattc?ctaa 24
<210>25
<211>16
<212>DNA
＜213〉artificial sequence
<400>25
taatgcagct?aaaagc 16
<210>26
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<212>DNA
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<400>26
tttgtctact?actactgaat?ca 22
<210>27
<211>22
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<400>27
aacggccata?cctgctgtat?at 22
<210>28
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<212>DNA
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<400>28
taggtacaca?ggctagtag 19
<210>29
<211>24
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<400>29
gctacacagg?ctaatgaata?caca 24
<210>30
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<212>DNA
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cactgctgtt?actcaatctg 20
<210>31
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<212>DNA
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<400>31
gctacatctg?ctgct 15
<210>32
<211>27
<212>DNA
<213>Human?papillomavirus?type?6
<400>32
caattctgat?tataaagagt?acatgcg 27
<210>33
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<212>DNA
<213>Human?papillomavirus?type?11
<400>33
taattcagat?tataaggaat?a 21
<210>34
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<212>DNA
<213>Human?papillomavirus?type?16
<400>34
cagaaactac?atataaaaat?actaactt 28
<210>35
<211>22
<212>DNA
<213>Human?papillomavirus?type?18
<400>35
cctatgatgc?taccaaattt?aa 22
<210>36
<211>25
<212>DNA
<213>Human?papillomavirus?type?31
<400>36
aattgcaaac?agtgatacta?cattt 25
<210>37
<211>16
<212>DNA
<213>Human?papillomavirus?type?33
<400>37
acacaagtaa?ctagtg 16
<210>38
<211>17
<212>DNA
<213>Human?papillomavirus?type?35
<400>38
gacagtacat?ataaaaa 17
<210>39
<211>16
<212>DNA
<213>Human?papillomavirus?type?39
<400>39
tctacatatg?atcctt 16
<210>40
<211>28
<212>DNA
<213>Human?papillomavirus?type?40
<400>40
taacagtaat?ttcaaggaat?atttgcgt 28
<210>41
<211>23
<212>DNA
<213>Human?papillomavirus?type?42
<400>41
caacatctgg?tgatacatat?aca 23
<210>42
<211>25
<212>DNA
<213>Human?papillomavirus?type?43
<400>42
ctactgtgcc?cagtacatat?gacaa 25
<210>43
<211>27
<212>DNA
<213>Human?papillomavirus?type?44
<400>43
gtctacatat?actagtgaac?aatataa 27
<210>44
<211>19
<212>DNA
<213>Human?papillomavirus?type?45
<400>44
ccaagtacat?atgacccta 19
<210>45
<211>31
<212>DNA
<213>Human?papillomavirus?type?51
<400>45
ccaacattta?ctccaagtaa?ctttaagcaa?t 31
<210>46
<211>29
<212>DNA
<213>Human?papillomavirus?type?52
<400>46
gaaagcacat?ataaaaatga?aaattttaa 29
<210>47
<211>14
<212>DNA
<213>Human?papillomavirus?type?53
<400>47
cagtctatgt?ctac 14
<210>48
<211>13
<212>DNA
<213>Human?papillomavirus?type?54
<400>48
atagctatat?ctg 13
<210>49
<211>24
<212>DNA
<213>Human?papillomavims?type?56
<400>49
gcacgaaaaa?ttaatcagta?cctt 24
<210>50
<211>30
<212>DNA
<213>Human?papillomavirus?type?58
<400>50
aaggtacata?taaaaatgat?aattttaagg 30
<210>51
<211>25
<212>DNA
<213>Human?papillomavirus?type?59
<400>51
tgtatacaca?cctaccagtt?ttaaa 25
<210>52
<211>22
<212>DNA
<213>Human?papillomavirus?type?66
<400>52
acattaacta?aatatgatgc?cc 22
<210>53
<211>33
<212>DNA
<213>Human?papillomavirus?type?68
<400>53
gctgtaccaa?atatttatga?tcctaataaa?ttt 33
<210>54
<211>29
<212>DNA
<213>Human?papillomavirus?type?70
<400>54
agccctacaa?agtttaagga?atatactag 29
<210>55
<211>27
<212>DNA
<213>Human?papillomavirus?type?73
<400>55
ctctactaca?acgtatgcca?actctaa 27
<210>56
<211>24
<212>DNA
<213>Human?papillomavirus?mm
<400>56
tgcacaaaca?tttactccag?caaa 24
<210>57
<211>27
<212>DNA
<213>Human?papillomavirus?type?83
<400>57
gcctctaact?ttaaggaata?cctccgc 27
<210>58
<211>26
<212>DNA
<213>Human?papillomavirus?CP8304
<400>58
gcagaataca?aggcctctaa?ctttaa 26
Claims (3)
1. a HPV detects and classifying method, it is characterized in that may further comprise the steps:
(a) with the HPV lysate HPV in the biological sample is carried out cracking, separation and Extraction nucleic acid;
(b) with the following Auele Specific Primer HPVL1 nucleic acid fragment that from step (a) gained nucleic acid, increases:
G1：5’-CARGGACAIAAYAATGGYATTTGCTG-3’(SEQ?ID?No.1)
G2：5’-CARGGACATAAYAATGGYATTTGTTG-3’(SEQ?ID?No.2)
G3：5’-GAAAAAYAAACTGYAAATCATAYTCCTC-3’(SEQ?ID?No.3)
G4：5’-GAAAAAYAAACTGYAAATCATAYTCTTC-3’(SEQ?ID?No.4)
Wherein G3/G4 is the vitamin H labeled primer;
(c) the HPVL1 nucleic acid fragment that step (b) is obtained carries out the tetra-sodium sequencing reaction, the tetra-sodium sequencing primer be selected from following any one or a few:
HPV6s：5’-AACTACATCTTCCACATACAC-3’(SEQ?ID?No.5)
HPV11s：5’-GTCTAAATCTGCTACATACAC-3’(SEQ?ID?No.6)
HPV16s：5’-ATTATGTGCTGCCATATCTACTT-3’(SEQ?ID?No.7)
HPV18s：5’-ACAGTCTCCTGTACCTGGG-3’(SEQ?ID?No.8)
HPV31s：5’-CCAATATGTCTGTTTGTGCTGC-3’(SEQ?ID?No.9)
HPV33s：5’-GTACTAATATGACTTTATGC-3’(SEQ?ID?No.10)
HPV35s：5’-GTGTTCTGCTGTGTCTTCTAGT-3’(SEQ?ID?No.11)
HPV39s：5’-TATAGAGTCTTCCATACCT-3’(SEQ?ID?No.12)
HPV40s：5’-CCCCACACCAACCCCATATAA-3’(SEQ?ID?No.13)
HPV42s：5’-CATGACTTTGTGTGCCACTG-3’(SEQ?ID?No.14)
HPV43s：5’-CGTTATGTGCCTCTACTGACC-3’(SEQ?ID?No.15)
HPV44s：5’-CTACACAGTCCCCTCC-3’(SEQ?ID?No.16)
HPV45s：5’-CTCTACACAAAATCCTGTG-3’(SEQ?ID?No.17)
HPV51s：5’-CTGCTGCGGTTTCC-3’(SEQ?ID?No.18)
HPV52s：5’-TTTATGTGCTGAGGTTAAAAAG-3’(SEQ?ID?No.19)
HPV53s：5’-CGCAACCACACAGTCTATG-3’(SEQ?ID?No.20)
HPV54s：5’-ACAGCATCCACGCAGG-3’(SEQ?ID?No.21)
HPV56s：5’-CAGAACAGTTAAGTAAATATGAT-3’(SEQ?ID?No.22)
HPV58s：5’-CACTGAAGTAACTAAGG-3’(SEQ?ID?No.23)
HPV59s：5’-TTCTACTACTTCTTCTATTCCTAA-3’(SEQ?ID?No.24)
HPV66s：5’-TAATGCAGCTAAAAGC-3’(SEQ?ID?No.25)
HPV68as：5’-TTTGTCTACTACTACTGAATCA-3’(SEQ?ID?No.26)
HPV70s：5’-AACGGCCATACCTGCTGTAIAT-3’(SEQ?ID?No.27)
HPV73s：5’-TAGGTACACAGGCTAGTAG-3’(SEQ?ID?No.28)
HPV83s：5’-GCTACACAGGCTAATGAAIACACA-3’(SEQ?ID?No.29)
HPVmm4s：5’-CACTGCTGTTACTCAATCTG-3’(SEQ?ID?No.30)
HPVCP8304s：5’-GCTACATCTGCTGCT-3’(SEQ?ID?No.31)
(d) sequence and the HPV type sequence that the tetra-sodium order-checking is read compared, and judges HPV somatotype result.
2. HPV classifying method as claimed in claim 1 is characterized in that the HPV lysate composition described in the step (a) is: 20mmol/L Tris.HCL pH8.0,0.5%Triton-X-100,10%chelex-100.
3. HPV classifying method as claimed in claim 1 is characterized in that the described HPV type of step (d) sequence comprises:
HPV6：5’-CAATTCTGATTATAAAGAGTACATGCG-3’(SEQ?ID?No.32)
HPV11：5’-TAATTCAGATTATAAGGAATA-3’(SEQ?ID?No.33)
HPV16：5’-CAGAAACTACATATAAAAATACTAACTT-3’(SEQ?ID?No.34)
HPV18：5’-CCTATGATGCTACCAAATTTAA-3’(SEQ?ID?No.35)
HPV31：5’-AATTGCAAACAGTGATACTACATTT-3’(SEQ?ID?No.36)
HPV33：5’-ACACAAGTAACTAGTG-3’(SEQ?ID?No.37)
HPV35：5’-GACAGTACATATAAAAA-3’(SEQ?ID?No.38)
HPV39：5’-TCTACATATGATCCTT-3’(SEQ?ID?No.39)
HPV40：5’-IAACAGTAATTTCAAGGAATATTTGCGT-3’(SEQ?ID?No.40)
HPV42：5’-CAACATCTGGTGATACATATACA-3’(SEQ?ID?No.41)
HPV43：5’-CTACTGTGCCCAGTACATATGACAA-3’(SEQ?ID?No.42)
HPV44：5’-GTCTACALATACTAGTGAACAATATAA-3’(SEQ?ID?No.43)
HPV45：5’-CCAAGTACATATGACCCTA-3’(SEQ?ID?No.44)
HPV51：5’-CCAACATTTACTCCAAGTAACTTTAAGCAAT-3’(SEQ?ID?No.45)
HPV52：5’-GAAAGCACATATAAAAATGAAAATTTTAA-3’(SEQ?ID?No.46)
HPV53：5’-CAGTCTATGTCTAC-3’(SEQ?ID?No.47)
HPV54：5’-AIAGCTATATCTG-3’(SEQ?ID?No.48)
HPV56：5’-GCACGAAAAATTAATCAGTACCTT-3’(SEQ?ID?No.49)
HPV58：5’-AAGGIACATATAAAAATGATAATTTTAAGG-3’(SEQ?ID?No.50)
HPV59：5’-TGTATACACACCTACCAGTTTTAAA-3’(SEQ?ID?No.51)
HPV66：5’-ACATTAACTAAATATGATGCCC-3’(SEQ?ID?No.52)
HPV68a：5’-GCTGTACCAAATATTTATGATCCTAATAAATTT-3’(SEQ?IDNo.53)
HPV70：5’-AGCCCTACAAAGTTTAAGGAATATACTAG-3’(SEQ?ID?No.54)
HPV73：5’-CTCTACTACAACGTATGCCAACTCTAA-3’(SEQ?ID?No.55)
HPVmm4：5’-TGCACAAACATTTACTCCAGCAAA-3’(SEQ?ID?No.56)
HPV83：5’-GCCTCTAACTTTAAGGAATACCTCCGC-3’(SEQ?ID?No.57)
HPVCP8304：5’-GCAGAATACAAGGCCTCTAACTTTAA-3’(SEQ?ID?No.58)。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994647A (en) * | 2012-06-21 | 2013-03-27 | 宁波海尔施基因科技有限公司 | Typing and quantitative detection method and kit for human papilloma virus (HPV) |
| CN107022649A (en) * | 2017-04-24 | 2017-08-08 | 南京医科大学 | A kind of marker detection method for predicting cervical cancer patient existence |
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| CN113508182A (en) * | 2018-12-18 | 2021-10-15 | 雅培分子公司 | Assays for detecting Human Papilloma Virus (HPV) |
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| CN100582239C (en) * | 2005-10-10 | 2010-01-20 | 上海透景生命科技有限公司 | Human papillomavirus detection and parting reagent kit |
| CN101397590A (en) * | 2008-10-27 | 2009-04-01 | 杭州迪安医学检验中心有限公司 | Typing method for human papilloma virus gene |
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| CN102994647A (en) * | 2012-06-21 | 2013-03-27 | 宁波海尔施基因科技有限公司 | Typing and quantitative detection method and kit for human papilloma virus (HPV) |
| CN102994647B (en) * | 2012-06-21 | 2014-11-19 | 宁波海尔施基因科技有限公司 | Typing and quantitative detection method and kit for human papilloma virus (HPV) |
| CN107022649A (en) * | 2017-04-24 | 2017-08-08 | 南京医科大学 | A kind of marker detection method for predicting cervical cancer patient existence |
| CN113508182A (en) * | 2018-12-18 | 2021-10-15 | 雅培分子公司 | Assays for detecting Human Papilloma Virus (HPV) |
| CN113508182B (en) * | 2018-12-18 | 2024-03-08 | 雅培分子公司 | Assay for detection of Human Papillomavirus (HPV) |
| CN112195287A (en) * | 2020-11-12 | 2021-01-08 | 武汉凯德维斯生物技术有限公司 | Probe group for human papilloma virus HPV typing and integration detection and kit thereof |
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