CN1814796A - Method for detecting and typing 26 human papillomaviruses - Google Patents
Method for detecting and typing 26 human papillomaviruses Download PDFInfo
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Abstract
This invention relates to a suspension chip technology for the gene test to human papillomavirus used in testing and typing 26 kinds of papillomaviruses, in which, specific probes of which are crosslinked on 26 kinds of fluorescent microspheres to be reacted with the tested specimens then reacted with the report molecules labeled by the fluorescein to test the type specific nucleic acid of the virus by a fluorescent test device, which can test 26 kinds of ordinary HPV once and increases the test efficiency greatly and overcomes the shortcoming of missing testing the potential infections in the serology and immunity test method to realize early diagnosis to HPA diseases.
Description
Technical field
The invention belongs to biotechnology, relate to fields such as molecular biology and biological chemistry, especially relate to the suspending chip technology of human papillomavirus gene test, be applicable to clinical detection human papillomavirus and gene type, to instruct the preventing and controlling of cervical cancer.
Background technology
Human papillomavirus human beings'health as the serious threat of human disease microorganism, and existing at present more than 100 kind of other HPV of different shaped identified that veriform HPV can cause different diseases.HPV infects and to be proved the first cause that causes cervical cancer and precancerous lesion thereof by epidemiology and biology, to HPV detect and somatotype be a kind of effectively, cervical cancer examination means reliably.Because the HPV in-vitro multiplication is difficulty very, does not up to the present still have reliable serological typing method.Traditional HPV detection method mainly is by morphology and immunohistochemical methods method it to be detected, as the Pap smear method, the liquid matrix cells is learned and is checked, vaginoscopy, histopathological examination, Electronic Speculum direct viewing HPV virion, radioimmunoprecipitation detects HPV16 antibody horizontal in patient's serum, enzyme-linked immunosorbent assay (ELISA) detects HPV-E6 in patient's serum, E7 specific antibody etc.The sensitivity of these detection methods and specificity are all not ideal enough, have higher false positive rate and false negative rate, and can not carry out somatotype to HPV.Classifying method commonly used have PCR, in situ hybridization, line sample probe analysis, etc., they exist easy pollution, false positive rate height, sensitivity is not high, operation is loaded down with trivial details, to shortcomings such as polyinfection are difficult to judge, cost an arm and a leg, are difficult to apply.
Recently for over ten years, many scientific workers adopt the modern molecular biology technique method that HPV is detected in order to overcome the deficiency of traditional detection method, and these methods mainly comprise nucleic acid hybridization, polymerase chain reaction PCR method.The sensitivity that these methods have is not high, the poor specificity that has, and what have is more time-consuming, the easy generation cross infection that has, the false positive rate height, the operation that has is loaded down with trivial details and can not somatotype.
Along with the develop rapidly of Protocols in Molecular Biology, biochip technology has been widely used in the detection of pathogenic micro-organism, and is bringing into play enormous function.
Based on the principle of chip, it is the suspending chip technology of carrier with the microsphere that U.S. Luminex company has developed.This technology is to utilize microsphere as carrier, and fluorescence detector carries out high throughput assay as detection platform to nucleic acid and protein and other.Different microsphere kinds can mark on the different 2 kinds of fluorescence of ratio as the numbering of microballoon.When the microsphere of difference numbering passed through fluorescence detector successively, instrument was analyzed the ratio of detected 2 kinds of fluorescence, thereby judges which kind of microsphere it is.Therefore, different microspheres can be placed on and pass through fluorescence detector in the same pipe, judges its numbering by computer software according to the fluorescence ratio.Different probe on the microsphere mark of these different numberings just can detect different pathogenic agent.At present, luminex company can provide 100 kinds of microballoons to use for high throughput testing.
Summary of the invention
The present invention has selected for use luminex company that 26 kinds of microspheres in 100 kinds of microballoons can be provided.
The method that the present invention is used for 26 kinds of detection of Human papillomavirus and somatotype is: with HPV 6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV58, HPV59, HPV66, HPV68, the type specificity probe of these 26 kinds of human papillomaviruss of HPV73 and HPV82 is crosslinked on 26 kinds of fluorescent microspheres, after tested sample reaction, with fluorescein-labeled reporter molecules reaction, detect the type specificity nucleic acid of human papillomavirus by fluorescence detector again.
The above 26 kinds to go into papilloma type specificity probe sequence as follows:
Table 1 HPV specific probe sequence
| SEQ NO | Type | Sequence(5’→3’) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 | HPV6 HPV 11 HPV 16 HPV 18 HPV 26 HPV 31 HPV 33 HPV 34 HPV 35 HPV 39 HPV 40 HPV 42 HPV 43 HPV 44 HPV 45 HPV 51 HPV 52 HPV 53 HPV 54 HPV 56 HPV 58 | 5’-TTTTTTTTTTTTTTTTTTATCCGTAACTACATCTTCCACATACACCAA-3’ 5’-TTTTTTTTTTTTTTTTTTATCTGTGTCTAAATCTGCTACATACACTAA-3’ 5’-TTTTTTTTTTTTTTTTTTGTCATTATGTGCTGCCAIATCTACTTCAGA-3’ 5’-TTTTTTTTTTTTTTTTTTTGCTTCTACACAGTCTCCTGTACCTGGGCA-3’ 5’-TTTTTTTTTTTTTTTTTTAGTACATTATCTGCAGCATCTGCATCCACT-3’ 5’-TTTTTTTTTTTTTTTTTTTGTTTGTGCTGCAATTGCAAACAGTGATAC-3’ 5’-TTTTTTTTTTTTTTTTTTTTTATGCACACAAGTAACTAGTGACAGTAC-3’ 5’-TTTTTTTTTTTTTTTTTTTACACAATCCACAAGTACAAATGCACCATA-3’ 5’-TTTTTTTTTTTTTTTTTTGTCTGTGTGTTCTGCTGTGTCTTCTAGTGA-3’ 5’-TTTTTTTTTTTTTTTTTTTCTACCTCTATAGAGTCTTCCATACCTTCT-3’ 5’-TTTTTTTTTTTTTTTTTTGCTGCCACACAGTCCCCCACACCAACCCCA-3’ 5’-TTTTTTTTTTTTTTTTTTCTGCAACATCTGGTGATACATATACAGCTG-3’ 5’-TTTTTTTTTTTTTTTTTTTCTACTGACCCTACTGTGCCCAGTACATAT-3’ 5’-TTTTTTTTTTTTTTTTTTGCCACTACACAGTCCCCTCCGTCTACATAT-3’ 5’-TTTTTTTTTTTTTTTTTTACACAAAATCCTGTGCCAAGTACATATGAC-3’ 5’-TTTTTTTTTTTTTTTTTTAGCACTGCCACTGCTGCGGTTTCCCCAACA-3’ 5’-TTTTTTTTTTTTTTTTTTTGCTGAGGTTAAAAAGGAAAGCACATATAA-3’ 5’-TTTTTTTTTTTTTTTTTTGTCTATGTCTACATATAATTCAAAGCAAAT-3’ 5’-TTTTTTTTTTTTTTTTTTTACAGCATCCACGCAGGATAGCTTTAATAA-3’ 5’-TTTTTTTTTTTTTTTTTTGTACTGCTACAGAACAGTTAAGTAAATATG-3’ 5’-TTTTTTTTTTTTTTTTTTATTATGCACTGAAGTAACTAAGGAAGGTAC-3’ |
| 22 23 24 25 26 | HPV 59 HPV 66 HPV 68 HPV 73 HPV 82 | 5’-TTTTTTTTTTTTTTTTTTTCTACTACTGCTTCTATTCCTAATGTATAC-3’ 5’-TTTTTTTTTTTTTTTTTTTATTAATGCAGCTAAAAGCACATTAACTAA-3’ 5’-TTTTTTTTTTTTTTTTTTTCTACTACTACTGAATCAGCTGTACCAAAT-3’ 5’-TTTTTTTTTTTTTTTTTTGTGTAGGTACACAGGCTAGTAGCTCTACTA-3’ 5’-TTTTTTTTTTTTTTTTTTCGACTCGTGTTACTCAATCTGTTGCACAAA-3’ |
The above 26 kinds of human papilloma type specificity probe crosslinked on 26 kinds of fluorescent microspheres is: each probe links together by chemical crosslink reaction with corresponding fluorescent microsphere.
The key step of detection of the present invention and somatotype is:
(1) microsphere with the good probe of mark mixes with tested sample, makes it to combine with corresponding target molecular specificity in the tested sample;
(2) in step (1), add streptavidin-phycoerythrin mixture;
(3) reactant of above-mentioned steps detects through fluorescence detector, judges the nucleic acid content of different shaped human papillomavirus by detected fluorescence intensity.
The method of detection of the present invention and somatotype is to be used for detection of Human papillomavirus and classification diagnosis.
The inventive method further describes as follows:
1, (China) 5 ' end is an amino group to each synthetic probe, then is the spacerarm of 18 T, is the type specificity probe of 30bp then for Invitrogen, Shanghai.26 probes link together by chemical crosslink reaction with corresponding different microballoons respectively, and 2-8 ℃ keeps in Dark Place behind the mark.
2, the preparation of sample to be tested: after sample wash-out to be measured, washing, be resuspended in 150 μ l lysates, 56 ℃ of water-baths 1 hour, phenol-chloroform-primary isoamyl alcohol method and ethanol precipitation obtain the DNA that PCR uses.
3, the amplification of testing sample and mark: PCR reaction is employed is that the MY09/11 primer is right, and upstream primer MY11 is 5 '-GCMCAGGGWCATAAYAATGG-3 ', and downstream primer MY09 is 5 '--CGTCCMARRGGAWACTGATC-3 ' (M=A+C; R=A+G; W=A+T; Y=C+T), 5 ' the terminal modified vitamin H Biotin.The PCR reaction system of 50-μ l contains the template DNA of 5 μ l, the upstream primer MY11 of 0.1 μ M, the biotin labeled downstream primer Biotin-MY09 of 0.8 μ M, the dNTPs of 0.2mM, the Taq archaeal dna polymerase of 2.5U, 1 * reaction buffer, 4mM MgCl
2Behind the mixing, 95 ℃ of sex change 5 minutes are subsequently carried out 95 ℃ of sex change of 40 circulations 20 seconds, 55 ℃ of annealing 30 seconds, and 72 ℃ were extended 45 seconds, and last 72 ℃ were extended 5 minutes.
4, hybridization and last machine testing: 12 μ lPCR products or positive control or negative control add 5 * SSC hybridization solution (include each microballoon that is marked with the type specificity probe respectively 5000) of 38 μ l.95 ℃ of sex change 5 minutes, 55 ℃ of hybridization 15 minutes.After reaction finishes, twice of washing microballoon.Add 25 μ l phycoerythrin.Lucifuge room temperature reaction 5 minutes.At last, at Luminex
100Detect on the analyzer.
Reaction principle of the present invention is summarized as follows:
Fluorescently-labeled microsphere, probe molecule, analyte and reporter molecule are 4 main composition parts of this detection system.Reaction mainly comprises 3 key steps: (1) probe molecule fixing; (2) with the microsphere and the reaction of tested sample of the good probe of mark, probe can combine with the corresponding target molecular specificity, has the reporter molecule of reporting fluorescence and also combines with the molecules of interest specificity.(3) reactant detects through fluorescence detector.
The principle of its detection: make one microsphere by sense channel, and use two-color laser simultaneously 2 kinds of fluorescence on the microsphere and the report fluorescence on the reporter molecule to be detected, thereby determine the kind and the quantity of analyte.
After utilizing the different antibodies bag to be carried out the paper publishing of multiple analysis detection by different addresses microsphere, this technology has obtained application in some respects.As specific probe, 16 kinds of bacteriums are identified with 16 kinds of bacterial 16 S rDNA; With different probe 30 people's HLA-DPB1 fragment is carried out snp analysis, resultant result is in full accord with the result that order-checking obtains; From blood plasma, detect the antibody of 16 kind of plant anaphylactogens with 16 kinds of antigens; The monoclonal antibody of anti-IL-6, IL-8 is connected on the microsphere, detects IL-6 and IL-8.This technology will become compatibility antigen in pathogenic agent, cytokine, the tissue matching and autoimmune disorder high-throughput, fast, the important tool of sensitive check and analysis.
The application is applied to this technology the gene type of HPV first, and its positively effect is:
The research that at present relevant HPV detects typing chip is also few, and we are applied to biochip technology detection and the somatotype of HPV.The HPV of this research preparation detects and typing gene chip is that employing is the suspension gene chip of carrier, design chips number of probes according to actual needs based on microballoon.
The invention has the advantages that:
1, human papillomavirus gene chip, can one-time detection 26 kinds of common type HPV, improved the detection efficiency of HPV greatly, shorten detection time.
Whether it is simple to operate that 2, the HPV gene chip detects somatotype, detect not only and can exist HPV to infect in the judgement sample by once hybridizing, and can identify the infection that belongs to the sort of type, is convenient to clinical application.
3, the HPV gene chip is with strong points, HPV-DNA in the sample is directly detected, infect the latent period that can detect HPV, overcome serology and method for immunohistochemical detection and can produce the defective of omission to latent infection, thereby realization is to the early stage quick diagnosis of HPV disease.
4, HPV gene chip, susceptibility height, high specificity.Designed the HPV specific oligonucleotide probe by the gene order comparison, designed positive control, negative control and blank simultaneously, the quality of chip has been controlled, got rid of false positive, false negative, made detected result more reliable.
5, the HPV gene chip can detect multiple HPV type simultaneously, and the judgement that multiple HPV is infected comes into plain view, and has overcome other HPV detection methods and has been difficult to judge polyinfection or operate loaded down with trivial details shortcoming.
6, the detected result objectivity is strong, the reliability height.Present technique is after a plurality of microsphere fluorescent signals are detected separately, to carry out statistical study with supporting software, makes detected result more accurately, reliably, greatly reduces the artificial subjective factor in the analytical results deterministic process.
Embodiment
One, the making method of the gene chip of HPV is as follows:
The present invention has designed HPV 6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV58, HPV59, HPV66, HPV68, the type specificity probe of these 26 kinds of viruses of HPV73 and HPV82, and synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.(China) 5 ' end is an amino group to each synthetic probe, then is the type specificity probe of 48bp for Invitrogen, Shanghai.Probe sequence is seen the table 1 of summary of the invention part.
Each probe links together by crosslinking reaction with corresponding microballoon.
Specific practice is 5 * 10
6Individual carboxylated microballoon is suspended in 50 μ l 100mM 2-(N-morpholino) ethanesulfonic acid (MES), in the reaction solution of pH 4.5, the amidized probe molecule that adds 1nmol, linking agent N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide (EDC) (the Pierce Chemical that adds 25. μ g, Rockford, IL) after, lucifuge reaction 30 minutes, add 25 μ g linking agents (EDC) again, the lucifuge reaction is 30 minutes again.Reaction is washed once with 0.02% Tween-20 liquid after finishing, and is washing once with 0.1% SDS liquid.At last, the microballoon that is marked with probe is suspended in TE, pH 8.0 (10mMTris-HCl, 1mM EDTA), 2-8 ℃ keeps in Dark Place.
Two, sample
(Hybrid Capture 2 HCII) detects 246 parts of clinical Uterine neck bush sample standard deviations, and wherein 140 parts have been considered to high-risk HPV infection, and other 106 parts of detected results are negative through the hybrid capture method.
Three, experimental technique
(1) extracts tissue sample DNA to be measured
PBS liquid with pH 7.2 elutes cervical exfoliated cell from the concussion of cell brush, PBS liquid is washed 2 times again.Cell is resuspended in 56 ℃ of water-baths of 150 μ l lysates (pH 8.0,10mM Tris-HCl, 1mM EDTA, 10mM NaCl, 0.5% SDS and 200 μ g/ml Proteinase Ks) 1 hour, 15 minutes inactivated proteases K of 95 ℃ of water-baths.Then, obtain the DNA that PCR uses by phenol-chloroform-primary isoamyl alcohol method and ethanol precipitation (method is with reference to " molecular cloning experiment guide " 98 editions).
(2) amplification of testing sample and mark
The MY-PCR reaction is employed to be that the MY09/11 primer is right, and upstream primer MY11 is 5 '-GCMCAGGGWCATAAYAATGG-3 ', and downstream primer MY09 is 5 '-Bitoin-CGTCCMARRGGAWACTGATC-3 ' (M=A+C; R=A+G; W=A+T; Y=C+T).The PCR reaction system of 50-μ l contains the template DNA of 5 μ l, 0.1 the upstream primer MY11 of μ M, 0.8 the biotin labeled downstream primer Biotin-MY09 of μ M, 0.2mM deoxynucleoside triphosphates (dNTPs), 2.5U the Taq archaeal dna polymerase (Promega, Shanghai, China), 1 * reaction buffer, 4mM MgCl
2Behind the mixing, put into PE9600PCR instrument (Perkin-Elmer, Foster City, Calif.) increase, reaction conditions is: 95 ℃ of sex change 5 minutes, subsequently carry out 95 ℃ of sex change of 40 circulations 20 seconds, annealed 30 seconds for 55 ℃, 72 ℃ were extended 45 seconds, extended 5 minutes down at 72 ℃ at last.The applied sample amount of 5 μ l carries out 1.5% agarose gel electrophoresis (method is with reference to " molecular cloning experiment guide " 98 editions).
What the gene amplification of β-globin was used is that the GH20/PC04 primer is right, and primer GH20 is 5 '-GAAGAGCCAAGGACAGGTAC-3 ', and primer PC04 is 5 '-CAACTTCATCCACGTTCACC-3 '.The same MY-PCR of the reaction system of the gene amplification of β-globin and amplification method, but primer is replaced by the GH20 of 0.2 μ M and the PC04 of 0.2 μ M respectively.
In order to verify the specificity of HPV suspension chip system, we are with biotin labeled and HPV type specificity probe sequence complementary artificial synthetic oligonucleotide with 26 kinds, hybridize with the HPV suspension chip system respectively.
(3) hybridization (lucifuge operation) and last machine testing
0.2-ml thin-walled PCR pipe in, add 5 * SSC hybridization solution (include each microballoon that is marked with the type specificity probe each 5000) of 38 μ l, add 12 μ l positive controls or negative control or PCR product (cumulative volume 50 μ l).On PE 9600PCR instrument, 95 ℃ of sex change 5 minutes, 55 ℃ of hybridization 15 minutes.After reaction finishes, on 96-well microtiter plates (Millipore Corporation, Bedford, MA 01730 U.S.A), twice of 100 μ l, 2 * SSC/0.02% Tween-20 liquid washing microballoon.At last, will be resuspended in 75 μ l, 2 * SSC/0.02% Tween-20 liquid.Add 25 μ l streptavidin-phycoerythrin mixtures (Streptavidin-R-phycoerythrin, 10 μ g/ml are dissolved in 2 * SSC/0.02% Tween-20).Lucifuge room temperature reaction 5 minutes.At last, at Luminex
100Detect on the analyzer.
(4) E7 type specificity PCR dna sequence dna is identified
E7 type specificity PCR can be to HPV16, and the high-risk HPV of 14 types such as 18,31,33,35,39,45,51,52,56,58,59,66 and 68 detect, and amplimer and amplification method are reported with reference to Walboomers etc.
(5) dna sequence dna is identified
E7 type specificity PCR only can detect 14 kinds of high-risk HPV.In the time of can not being detected by E7 type specificity PCR if sample can be increased by MY-PCR, just to the evaluation of checking order of this MY-PCR product.The MY-PCR amplified production carries out dna sequence dna in Megabase 500 automated DNA analytical systems and identifies behind QIAquick PCR Purification kit purifying.If suspect when the MY-PCR amplified production contains HPV dna sequence dna more than two types, earlier the MY-PCR product is carried out the TA clone, then the single clone of picking carries out dna sequence dna respectively and identifies.The sequence that order-checking obtains is carried out the BLAST analysis respectively on the net, in order to determine the being HPV (www.ncbi.nlm.nih.gov) of that type.
Four, result
(1) PCR Quality Control
In the time of the MY-PCR amplification, need carry out the amplification of β-globin gene, monitor whether there is the PCR inhibition with this.The sample that does not particularly have the MY-PCR amplified production must carry out the amplification of β-globin gene, to be confirmed to be true negative or false negative.
(2) specificity of HPV Gene Chip system and limit of detection
Be with biotin labeled and HPV type specificity probe sequence complementary artificial synthetic oligonucleotide, hybridize with the HPV Gene Chip system respectively for 26 kinds.The result shows, every kind of microballoon and corresponding artificial synthetic oligonucleotide's (2nmol/L) hybridization signal is all greater than 2000MIF, and the HPV Gene Chip system has good signal-noise ratio, and finds no cross reaction.
In order to assess the limit of detection of this HPV suspension chip system, we carry out gradient dilution to the artificial synthetic oligonucleotide of HPV16 type, hybridize with this detection system respectively again.The result shows, under our experiment condition, as long as contain 1.024 * 10 in the hybridization system of 50 μ l
14During with last copy number, just can be detected next by this system.
(3) the HPV Gene Chip system is to the detection and the somatotype of clinical sample
1, MY-PCR result:
246 parts of clinical samples all pass through Hybrid Capture 2 (HCII) and detect, and wherein 140 parts have been considered to high-risk HPV infection, and other 106 parts of detected results are negative.130 parts of amplified productions that can obtain MY-PCR in 140 parts of positive sample.3 parts of amplified productions that also obtain MY-PCR in 106 parts of negative sample, totally 133 these MY-PCR of increment positive as a result.
2, HPV suspending chip detected result:
The HPV suspension chip system detects 133 parts of MY-PCR amplified productions, the results are shown in Table 1.There are 12 parts in 133 parts of MY-PCR amplified productions and fail to be detected, the results are shown in Table 1 by the HPV Gene Chip system.These 12 parts of MY-PCR amplified productions are carried out determined dna sequence, and the result confirms that these 12 parts of HPV types that product contained are not included in 26 kinds of HPV types that this HPV suspension chip system can detect.
121 parts of MY-PCR amplified productions that detect through the HPV Gene Chip system also carry out E7 type specificity PCR detection or dna sequence dna simultaneously and identify.The result that E7 type specificity PCR detects or dna sequencing is identified shows that it is correct that the HPV Gene Chip system detects the result that 120 parts of MY-PCR amplified productions are detected.Have only the result that the detected result of 1 part of MY-PCR amplified production and E7 type specificity PCR detect or DNA identifies inconsistent.The result that the HPV Gene Chip system detects shows that this sample infects 3 kinds of HPV, is respectively HPV31, HPV66 and HPV73.HPV31 and HPV66 can be detected by E7 type specificity PCR, but E7 type specificity PCR and dna sequencing all fail to detect HPV73.This problem need be done further solution.
Above-mentioned 133 increments detected result was originally all carried out 3 times and was repeated, and the result of 3 detections is consistent.From The above results, the detected result of this HPV Gene Chip system is consistent with the E7 type specificity PCR of routine or the result of dna sequencing, and the detected result that shows this HPV Gene Chip system is reliable.
Table 1 MY-PCR, E7 type specificity PCR (or dna sequence dna evaluation) detected result and the inspection of HPV Gene Chip system
Survey result's comparison
| HPV gene chip sample (n) | MY-PCR | E7 type specificity PCR or dna sequencing | ||||
| + | - | Amount to | + | - | Amount to | |
| +-amount to | 121 12 133 | 0 113 113 | 121 125 246 | 120 0 120 | 1 12 13 | 121 12 133 |
No difference of science of statistics between HPV Gene Chip system detected result and E7 type specificity PCR or the dna sequencing detected result.(McNemar ' s test value＞0.9)
Claims (5)
1. method that is used for 26 kinds of detection of Human papillomavirus and somatotype is characterized in that: with HPV6, and HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV58, HPV59, HPV66, HPV68, the type specificity probe of these 26 kinds of human papillomaviruss of HPV73 and HPV82 is crosslinked on 26 kinds of fluorescent microspheres, after tested sample reaction, with fluorescein-labeled reporter molecules reaction, detect the type specificity nucleic acid of human papillomavirus by fluorescence detector again.
2. by the described method that is used for 26 kinds of detection of Human papillomavirus and somatotype of claim 1, it is characterized in that: described 26 kinds of human papilloma type specificity probe sequences are as follows:
SEQ NO Type Sequence(5’→3’)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 HPV6 HPV 11 HPV 16 HPV 18 HPV 26 HPV 31 HPV 33 HPV 34 HPV 35 HPV 39 HPV 40 HPV 42 HPV 43 HPV 44 HPV 45 HPV 51 HPV 52 HPV 53 HPV 54 HPV 56 5’-TTTTTTTTTTTTTTTTTTATCCGTAACTACATCTTCCACATACACCAA-3’ 5’-TTTTTTTTTTTTTTTTTTATCTGTGTCTAAATCTGCTACATACACTAA-3’ 5’-TTTTTTTTTTTTTTTTTTGTCATTATGTGCTGCCATATCTACTTCAGA-3’ 5’-TTTTTTTTTTTTTTTTTTTGCTTCTACACAGTCTCCTGTACCTGGGCA-3’ 5’-TTTTTTTTTTTTTTTTTTAGTACATTATCTGCAGCATCTGCATCCACT-3’ 5’-TTTTTTTTTTTTTTTTTTTGTTTGTGCTGCAATTGCAAACAGTGATAC-3’ 5’-TTTTTTTTTTTTTTTTTTTTTATGCACACAAGTAACTAGTGACAGTAC-3’ 5’-TTTTTTTTTTTTTTTTTTTACACAATCCACAAGTACAAATGCACCATA-3’ 5’-TTTTTTTTTTTTTTTTTTGTCTGTGTGTTCTGCTGTGTCTTCTAGTGA-3’ 5’-TTTTTTTTTTTTTTTTTTTCTACCTCTATAGAGTCTTCCATACCTTCT-3’ 5’-TTTTTTTTTTTTTTTTTTGCTGCCACACAGTCCCCCACACCAACCCCA-3’ 5’-TTTTTTTTTTTTTTTTTTCTGCAACATCTGGTGATACATATACAGCTG-3’ 5’-TTTTTTTTTTTTTTTTTTTCTACTGACCCTACTGTGCCCAGTACATAT-3’ 5’-TTTTTTTTTTTTTTTTTTGCCACTACACAGTCCCCTCCGTCTACATAT-3’ 5’-TTTTTTTTTTTTTTTTTTACACAAAATCCTGTGCCAAGTACATATGAC-3’ 5’-TTTTTTTTTTTTTTTTTTAGCACTGCCACTGCTGCGGTTTCCCCAACA-3’ 5’-TTTTTTTTTTTTTTTTTTTGCTGAGGTTAAAAAGGAAAGCACATATAA-3’ 5’-TTTTTTTTTTTTTTTTTTGTCTATGTCTACATATAATTCAAAGCAAAT-3’ 5’-TTTTTTTTTTTTTTTTTTTACAGCATCCACGCAGGATAGCTTTAATAA-3’ 5’-TTTTTTTTTTTTTTTTTTGTACTGCTACAGAACAGTTAAGTAAATATG-3’
3. by the described method that is used for 26 kinds of detection of Human papillomavirus and somatotype of claim 1, it is characterized in that: described 26 kinds of human papilloma type specificity probes crosslinked on 26 kinds of fluorescent microspheres is: each probe links together by chemical crosslink reaction with corresponding fluorescent microsphere.
4. by the described method that is used for 26 kinds of detection of Human papillomavirus and somatotype of claim 1, it is characterized in that the key step that detects with somatotype is:
(1) microsphere with the good probe of mark mixes with tested sample, makes it to combine with corresponding target molecular specificity in the tested sample;
(2) in step (1), add streptavidin-phycoerythrin mixture;
(3) reactant of above-mentioned steps detects through fluorescence detector, judges the nucleic acid content of different shaped human papillomavirus by detected fluorescence intensity.
5. by the described method that is used for 26 kinds of detection of Human papillomavirus and somatotype of claim 1, it is characterized in that: this method is used for detection of Human papillomavirus and classification diagnosis.
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