CN1687451A - Diagnosis chip for testing subtype of human papilomavirus gene and preparation method and test method - Google Patents

Diagnosis chip for testing subtype of human papilomavirus gene and preparation method and test method Download PDF

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CN1687451A
CN1687451A CN 200510013318 CN200510013318A CN1687451A CN 1687451 A CN1687451 A CN 1687451A CN 200510013318 CN200510013318 CN 200510013318 CN 200510013318 A CN200510013318 A CN 200510013318A CN 1687451 A CN1687451 A CN 1687451A
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hpv
chip
probe
point
genotype
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CN100400675C (en
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高英堂
方淑昌
杜智
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ZIBO HIGH TECH Co Ltd TIANJIN CITY
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ZIBO HIGH TECH Co Ltd TIANJIN CITY
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Abstract

This invention is a method of preparing and detecting diagnostic chip of human papillomavirus gene. On the base of the chip there are 16 kinds of stylet which arrange in array, they consist of one general stylet, 15stylet subjected to HPV. There are 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 52, 56, 58 and so on 15 in total. We add a nucleic acid to the end of the atyle, irradiate the chip under the ultraviolet radiation for 5minute, fix the aryle to the surface of the chip, and construct to be diagnostic chip. PCR primer adapts 2-24 chain of primer. This invention has the virtue of quick, exactness, simple and convenient, special recognition, high information, it is prone to expand, and has high economic and social efficiency on clinic application.

Description

Diagnosis chip for testing subtype of human papilomavirus gene and making method and detection method
Technical field
The present invention relates to a kind of microorganism detection medical science vitro diagnostic techniques, relate in particular to a kind of diagnosis chip for testing subtype of human papilomavirus gene and making method and detection method.
Background technology
The somatotype research of human papillomavirus (being Human papilloma virus, hereinafter to be referred as HPV) is subjected to the great attention of Chinese scholars always, and method therefor is depended in the various affirmation in sample of HPV.At present, the HPV diagnosis of infection is mainly relied on Protocols in Molecular Biology viral nucleotide sequences is carried out gene test, comprise that methods such as gene sequencing, hybrid capture, dot hybridization, slot hybridization and Idiotype probe have been applied to HPV research, but the HPV virogene type that because separately limitation and particular requirement, can't satisfy in clinical detection in the short period of time, high-throughput obtains the patient infection.
Summary of the invention
Main purpose of the present invention is to solve the problem that above-mentioned detection HPV virogene exists, and a kind of quick, reliable, responsive, the special and simple diagnosis chip for testing subtype of human papilomavirus gene of biochip technology and making method and detection method utilized is provided.
Human papillomavirus (being Human papilloma virus, hereinafter to be referred as HPV) is a kind of dna virus of very easily propagating diffusion in the crowd, can be by directly or indirectly contact cross infection, and its infection site is hidden, and the morbidity concealment is difficult for early discovery.Show that according to the relevent statistics nearly 20% crowd is carrying HPV virus, and do not have any symptom above the people of half after infecting HPV, therefore, the population ratio of the actual HPV of carrying may be far above in this.Each genotype of HPV and its biological behaviour exist high correlation, and the HPV of different genotype can cause the differential responses and the disease of human body different sites, and different pathogenic hazardness is arranged.The skin and the mucomembranous epithelial cell of HPV infected person bring out hyperplasia, produce nipple sample pathology, and the infection that different genotype causes has different clinical manifestations.
The difference that is played a role in genital system tumor forms according to HPV is divided into HPV high-risk group, middle danger group and hangs down three groups of danger group.Clinical statistics shows, high-risk group HPV infect be cervical cancer main diseases because of, fall ill relevant with the cervical cancer 95% or more.If can find HPV early, treat indirectly, can significantly reduce the chance of suffering from cervical cancer with medicine.Even unfortunate discovery suffers from cervical cancer, as long as can find and treat in first to the second phase that the uterine neck carninomatosis is sent out, survival rate reaches 70 to 90% in 5 years; If but were later than the discovery of the 3rd to the fourth phase, survival rate would have only 7-30% in its 5 years.Therefore carry out the HPV gene type early, very necessary to women's health.In addition, document shows that HPV also exists in the squamous cell mucomembranous tumour of reproductive system with exterior domain recently, as oral cavity, nasopharynx, esophagus and male urethra etc.
The technical solution adopted for the present invention to solve the technical problems is:
Probe is set on the chip matrix, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of at least 5 above coordinate point control chips, with the rectangle point sample of coordinate point control chip zone and point quadrat to, an angle is provided with 2 punctuates in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, more than file at least 3 row of microarray, the walking crosswise more than at least 3 row of microarray.Each HPV probe point sample is more than 1, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represent 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, the dot matrix number is more than at least 18.
The PCR primer adopts the nested primer combination of 2 pairs of 24 chains, and its nucleotide sequence is as shown in the table,
Genotype Sequence is formed
The upstream outer primer
????6/11 ????5’GC(C/T)CAGGGACATAACAATGG3′
????18/58 ????5’GCACA(G/A)GGTCATAACAATGG3′
????39/45 ????5’GCCCAGGGCCA(T/C)AACAATGG3′
????33/35/56 ????5’GCACAAGG(T/C)CATAATAATGG3′
????31/42 ????5’GCTCA(G/A)GGACACAATAATGG3′
????44/52/16 ????5’GC(G/A)CAGGGCCACAATAATGG3′
The upstream inner primer
????6/11/18/31/33 ????5’TTTGTTACTGTGGTAGATAC3′
????44/56 ????5’TTTGTTACTGT(T/A)GTAGATAC3′
????35/58/42 ????5’TTTGTTAC(T/C)GT(A/G)GTTGATAC3′
????52 ????5’TTTGTCACAGTTGTGGATAC3′
????39/45 ????5’TTTGTTACTGT(A/T)GTGGACAC3′
????16 ????5’TTTGTTACTGTTGTTGATAC3′
The downstream outer primer
????6/33/58 ????5’CGTCCCAAAGGA(T/A)ACTGATC3′
????35 ????5’CGGCCCAACGGAAACTGATC3′
????31/56 ????5’CGACCCAGTGGAAA(C/T)TGATC3′
????39/42 ????5’CGTCC(T/C)AAAGG(A/G)AATTGATC3′
????11/18/44/45 ????5’CGTCCAAGGGGA(A/T)A(C/T)TGATC3′
????16/52 ????5’CGTCCTAAAGGAAACTGATC3′
The downstream inner primer
????6/52/31/39 ????5’TGAAA(A/T)ATAAATTGTAAATCA3′
????33/58 ????5’TGAAAAACAAACTGTA(G/A)(A/G)TCA3′
????44 ????5’TGAAACATAAATTGTAAGTCA3′
????56 ????5’TGAAAAACAAATTGTAATTCA3′
????42 ????5’TGAAATATAAATTGCACATCA3′
????18/45/35/11/16 ????5’TGAAAAATAAACTG(C/T)AAATCA3′
Fixing each genotype probe of HPV on positively charged chip matrix, probe are the two point microarray and arrange on film.
The point sample of 5 coordinate point (black circles) control chip zone and point quadrat to, two coordinate point in the lower left corner are the point sample starting point.Each HPV probe can repeat 2 of point samples, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56 represent 15 kinds of genotype, and "+" represents positive quality control point.
1 of the genotypic general probe of HPV, 15 of HPV genotype specific probes, comprise that HPV genotype HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 etc. amount to 15 kinds of genotype, various probe sequence is as shown in the table
Genotype Sequence is formed
General probe ????5′TATGATTT(A/G)CA(A/G)TTTATG3′
????HPV-6 ????5’TCCGTAACTACATCTTCCA3′
????HPV-11 ????5’TCTGTGTCTAAATCTGCTAC3′
????HPV-16 ????5’CATTATGTGCTGCCATATC3′
????HPV-18 ????5’TGCTTCTACACAGTCTCCT3′
????HPV-31 ????5’GCAATTGCAAACAGTGATAC3′
????HPV-33 ????5’TATGACTTTATGCACACAAGT3′
????HPV-35 ????5’CTGCTGTGTCTTCTAGTGA3′
????HPV-39 ????5’ATAGAGTCTTCCATACCTTC3′
????HPV-42 ????5’TGGTGATACATATACAGCTG3′
????HPV-43 ????5’TCTACTGACCCTACTGTG3′
????HPV-44 ????5’TACTAGTGAACAATATAAGCA3′
????HPV-45 ????5’TACACAAAATCCTGTGCCA3′
????HPV-52 ????5’GAATACCTTCGTCATGGC3′
????HPV-56 ????5’CAGAACAGTTAAGTAAATATG3′
????HPV-58 ????5’ATTATGCACTGAAGTAACTAA3′
15 HPV type specific probes refer to distinguishes 15 kinds of genotypic type specific probes of HPV, a kind of genotype that each probe is corresponding specific.1 total probe refers to 15 kinds of genotypic general probes of HPV, and this probe can hybridization signal occur with 15 kinds of HPV genotype, even comprises other genotype.
Add one section specific polynucleotide tail at the specific probe end, it can be solidificated on the chip, specific polynucleotide tail is 20 the deoxythymidine acid molecules of 3 ' terminal adding at each HPV specific probe.The chip that point is good is placed on and shone in the UV-crosslinked instrument 5 minutes, and probe is completely fixed on the chip, constitutes and detects diagnosing chip, the detection diagnosing chip that constitutes is placed under the 2-8 ℃ of condition deposit.
Each genotype sequence of HPV according to American National bioinformation center gene pool (GenBank) announcement, choose the ORF L1 district of guarding and exist the type differences in the HPV sequence relatively, detect genotypic PCR primer and chip probe in conjunction with each genotype of HPV at the popular situation and the pathogenic designing institute of China, add one section specific polynucleotide tail at 3 ' end when probe is synthetic.
Probe is set on chip matrix, place point sample probe and coordinate mark with microwell plate, probe rectangle microarray on chip is arranged, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58,58 amount to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of at least 5 above coordinate point control chips, with coordinate point indicate the rectangle point sample zone of control chip and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, more than file at least 3 row of microarray, the walking crosswise more than at least 3 row of microarray.Each HPV probe can repeat 2 of point samples, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56 represent 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, the dot matrix number is more than at least 18.
The PCR primer adopts the nested primer combination of 2 pairs of 24 chains, and its nucleotide sequence is as shown in the table,
Genotype Sequence is formed
The upstream outer primer
????6/11 ????5’GC(C/T)CAGGGACATAACAATGG3′
????18/58 ????5’GCACA(G/A)GGTCATAACAATGG3′
????39/45 ????5’GCCCAGGGCCA(T/C)AACAATGG3′
????33/35/56 ????5’GCACAAGG(T/C)CATAATAATGG3′
????31/42 ????5’GCTCA(G/A)GGACACAATAATGG3′
????44/52/16 ????5’GC(G/A)CAGGGCCACAATAATGG3′
The upstream inner primer
????6/11/18/31/33 ????5’TTTGTTACTGTGGTAGATAC3′
????44/56 ????5’TTTGTTACTGT(T/A)GTAGATAC3′
????35/58/42 ????5’TTTGTTAC(T/C)GT(A/G)GTTGATAC3′
????52 ????5’TTTGTCACAGTTGTGGATAC3′
????39/45 ????5’TTTGTTACTGT(A/T)GTGGACAC3′
????16 ????5’TTTGTTACTGTTGTTGATAC3′
The downstream outer primer
????6/33/58 ????5’CGTCCCAAAGGA(T/A)ACTGATC3′
????35 ????5’CGGCCCAACGGAAACTGATC3′
????31/56 ????5’CGACCCAGTGGAAA(C/T)TGATC3′
????39/42 ????5’CGTCC(T/C)AAAGG(A/G)AATTGATC3′
????11/18/44/45 ????5’CGTCCAAGGGGA(A/T)A(C/T)TGATC3′
????16/52 ????5’CGTCCTAAAGGAAACTGATC3′
The downstream inner primer
????6/52/31/39 ????5’TGAAA(A/T)ATAAATTGTAAATCA3′
????33/58 ????5’TGAAAAACAAACTGTA(G/A)(A/G)TCA3′
????44 ????5’TGAAACATAAATTGTAAGTCA3′
????56 ????5’TGAAAAACAAATTGTAATTCA3′
????42 ????5’TGAAATATAAATTGCACATCA3′
????18/45/35/11/16 ????5’TGAAAAATAAAC?TG(C/T)AAATCA3′
1 of the genotypic general probe of HPV, 15 of HPV genotype specific probes, comprise that HPV genotype HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52,5HPV6, HPV58 amount to 15 kinds of genotype, various probe sequence is as shown in the table
Genotype Sequence is formed
General probe ????5′TATGATTT(A/G)CA(A/G)TTTATG3′
????HPV-6 ????5’TCCGTAACTACATCTTCCA3′
????HPV-11 ????5’TCTGTGTCTAAATCTGCTAC3′
????HPV-16 ????5’CATTATGTGCTGCCATATC3′
????HPV-18 ????5’TGCTTCTACACAGTCTCCT3′
????HPV-31 ????5’GCAATTGCAAACAGTGATAC3′
????HPV-33 ????5’TATGACTTTATGCACACAAGT3′
????HPV-35 ????5’CTGCTGTGTCTTCTAGTGA3′
????HPV-39 ????5’ATAGAGTCTTCCATACCTTC3′
????HPV-42 ????5’TGGTGATACATATACAGCTG3′
????HPV-43 ????5’TCTACTGACCCTACTGTG3′
????HPV-44 ????5’TACTAGTGAACAATATAAGCA3′
????HPV-45 ????5’TACACAAAATCCTGTGCCA3′
????HPV-52 ????5’GAATACCTTCGTCATGGC3′
????HPV-56 ????5’CAGAACAGTTAAGTAAATATG3′
????HPV-58 ????5’ATTATGCACTGAAGTAACTAA3′
Use the dna synthesizer synthesising probing needle, add one section specific polynucleotide tail at the specific probe end, i.e. 20 oligomerization thymidylic acids, it can be solidificated on the chip, adopt the solid phase phosphoramidite triester method to add 20 deoxythymidine acid molecules at 3 of each HPV specific probe ' end at dna synthesizer.Use the Nunc point sample instrument to carry out point sample at the chip that has positive charge according to pre-set order.
The chip that has positive charge that uses Roche Holding Ag to produce uses the Nunc point sample instrument to carry out point sample by pre-set order.
The chip that point is good is placed on the UV-crosslinked instrument internal radiation of UVP and fixes 5 minutes, makes the point sample probe stationary on chip, constitute to detect diagnosing chip, the detection diagnosing chip that is made into place deposit under the 2-8 ℃ of condition stand-by.
Because designed probe is shorter, be difficult to be solidificated on the chip, therefore 3 ' end at probe sequence adds a tail, and promptly 20 oligomerization thymidylic acids can be solidificated on the chip it.
Design HPV Auele Specific Primer and probe:, adopt the combination nested primer design of 2 pairs of 24 chains for effectively each genotype HPV of amplification and raising sensitivity.16 of designing probes comprise 1 of the shared probe of HPV, distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56 and HPV58 and amount to 15 kinds of genotype.
Take out the chip that obtains hybridization signal, whole chip background is not painted, and hybridization signal is pale brown look, and the difference between specific hybridization point and the non-specific hybridization point is obvious, and by point sample region mode figure, the HPV gene type is found in comparison.
Interpretation according to clinical samples processing, sleeve type PCR amplification, chip hybridization, chip washing, chip colour developing and chip hybridization signal detects; Carry out clinical samples and handle employing biopsy or paraffin specimen or swab specimen.
The sleeve type PCR amplification is divided into twice to be carried out, and pcr amplification comprises sample disposal supernatant liquor 5 microlitres, a PCR reaction mixture (containing built-up type upstream outer primer or downstream outer primer) 44 microlitres and Taq polysaccharase 1 microlitre for the first time, amounts to 50 microlitre uniform mixing.The PCR cycling condition is 94 ℃ of pre-sex change 2 minutes, 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 35 circulations, keep 72 ℃ to extend 5 minutes.
For the second time pcr amplification is with the first time PCR product 5 microlitres, secondary PCR reaction mixture (containing built-up type upstream inner primer and DIG-dUTP or downstream inner primer and DIG-dUTP) 44 microlitres and Taq polysaccharase 1 microlitre uniform mixing.
The PCR cycling condition is 94 ℃ of pre-sex change 2 minutes, 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 35 circulations, keep 72 ℃ to extend 5 minutes, promptly be made into the hybridization probe of DIG mark through the secondary PCR amplification PCR products.
The chip of making is in advance packed in the hybridization bag, and in hybridization bag, add an amount of hybridization solution, hybridization bag is sealed, carry out 0.5-1 hour prehybridization, add the sex change probe of 5-20 microlitre, put into the hybridization case, carry out 3-6 hour hybridization again, hybridization oven temperature, degree is 45 ℃.
Use 2 * SSC/0.1%SDS washing chip twice at ambient temperature, each 2min, the chip of under 46 ℃ of conditions, washing twice with 0.2 * SSC/0.1%SDS, each 2min uses TNT solution washing chip 5min then.
The chip of washes clean is packed in the clean plastics bag, in plastics bag, add 10% blockade liquid 100 μ l or TN900 μ l, mix, under 37 ℃ condition, place 30min.
Add POD antibody 1 μ l, mix gently, under 37 ℃ condition, place 30min; Chip is taken out usefulness TNT rinsing chip 2 times from plastics bag, each 3min.
Chip is put into clean plastics bag, get DAB colour developing liquid 10 μ l, DAB diluent 10 μ l and aqua sterilisa 480 μ l, be added in the plastics bag immediately after mixing, treat that colour developing reaches (generally developing the color about 5-30 minute) after the desired level, with 1 * PBS termination reaction, obtain the chip hybridization result, whole chip background is not painted, and hybridization signal is pale brown look.
Clinical samples is handled and is adopted biopsy, get and grind after 50 milligrams of biopsies adding dna cleavage liquid 100 microlitres mix, carrying out 55 ℃ of temperature after the grinding bathed 1-3 hour, carrying out 100 ℃ again boiled 10 minutes, with the 12000rpm whizzer centrifugal 10 minutes, get 5 microlitre supernatant liquors and be used as the pcr amplification template.
Clinical samples is handled and is adopted paraffin specimen, gets 3-5 paraffin specimen section and dewaxes, and adds dna cleavage liquid 50 microlitres, carrying out 55 ℃ of temperature bathed 1-3 hour, carry out 100 ℃ again and boiled 10 minutes, centrifugal 10 minutes of the whizzer of usefulness 12000rpm is got 5 microlitre supernatant liquors and is used as the pcr amplification template.
Clinical samples is handled and is adopted swab specimen, swab specimen soaks with physiological saline, precipitate with centrifugal mode collecting cell, add dna cleavage liquid 50 microlitres, carrying out 55 ℃ of temperature bathed 1-3 hour, carry out 100 ℃ again and boiled 10 minutes, use centrifugal 10 minutes of 12000rpm whizzer, get 5 microlitre supernatant liquors and be used as the pcr amplification template.
The present invention carries out analysis revealed in No.3 Central Hospital of Tianjin City to the 95 routine clinical samples of collecting, and wherein 25 examples are the positive infection of HPV, and the somatotype result is as follows:
Mixed type 1 example of mixed type 1 example, HPV16 and the HPV52 of HPV6 type 9 examples, HPV11 type 5 examples, HPV16 type 4 examples, HPV33 type 3 examples, HPV52 type 2 examples, HPV16 and HPV33, chip hybridization the results are shown in Figure 2.The PCR product of wherein 25 examples is carried out further cloning and sequencing, all confirm the accuracy of chip.Experimental results show that the HPV genotyping diagnosis chip have diagnosis accurately, high specificity, simple to operate, the characteristics that contain much information.
The present invention is diagnosis chip for testing subtype of human papilomavirus gene and making method and detection method.Have diagnosis accurately, fast, simple, the high specificity of diagnostic method, quantity of information is high and the characteristics that are easy to promote the use of, the probe in detecting flux is big, can detect 15 kinds of common HPV genotype simultaneously; Easy and simple to handle, quick, do not need expensive plant and instrument and strict operating environment, begin only with obtaining detected result in two working dayss from sample process; The built-up type design of primers has improved the efficient and the positive rate of pcr amplification; Reasonably probe design has reduced the generation of non-specific hybridization reaction, high specificity; Detected result is easy to judge, can directly judge by naked eyes, also can be by the automatic Collection and analysis data of computer-controlled chip reading apparatus.The market potential of clinical application is huge, has great economic benefit and social benefit.
Description of drawings
Below in conjunction with drawings and Examples to the detailed description of the invention.
Fig. 1 chip micro probe array spot sample mode figure (A) 7 * 4 microarray patterns (B) 9 * 3 microarray patterns (C) 5 * 6 microarray patterns (D) 4 * 7 microarray patterns (E) 3 * 12 microarray patterns
The genotypic chip hybridization result of Fig. 2 section H PV (A) HPV6 type (B) HPV11 type (C) HPV16 type (D) HPV33 type (E) HPV16,33 mixed types (F) HPV16,52 mixed types (annotate: the pencil word is mark this shop among the figure)
Embodiment
Embodiment 1
Probe is set on the chip matrix, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of 5 coordinate point control chips, with the rectangle point sample of coordinate point control chip zone and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, the file of microarray is 4 row, and walking crosswise of microarray is 7 row.Each HPV35, HPV42, HPV43, HPV45, HPV56, the HPV58 probe points is 1, general probe, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV44, the HPV52 probe points is 2, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represents 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, several 18 points of dot matrix.
The PCR primer adopts the nested primer combination of 2 pairs of 24 chains, and its nucleotide sequence is as shown in the table,
Genotype Sequence is formed
The upstream outer primer
????6/11 ????5’GC(C/T)CAGGGACATAACAATGG3′
????18/58 ????5’GCACA(G/A)GGTCATAACAATGG3＇
????39/45 ????5’GCCCAGGGCCA(T/C)AACAATGG3′
????33/35/56 ????5’GCACAAGG(T/C)CATAATAATGG3′
????31/42 ????5’GCTCA(G/A)GGACACAATAATGG3′
????44/52/16 ????5’GC(G/A)CAGGGCCACAATAATGG3′
The upstream inner primer
????6/11/18/31/33 ????5’TTTGTTACTGTGGTAGATAC3′
????44/56 ????5’TTTGTTACTGT(T/A)GTAGATAC3′
????35/58/42 ????5’TTTGTTAC(T/C)GT(A/G)GTTGATAC3′
????52 ????5’TTTGTCACAGTTGTGGATAC3′
????39/45 ????5’TTTGTTACTGT(A/T)GTGGACAC3′
????16 ????5’TTTGTTACTGTTGTTGATAC3′
The downstream outer primer
????6/33/58 ????5’CGTCCCAAAGGA(T/A)ACTGATC3′
????35 ????5’CGGCCCAACGGAAACTGATC3′
????31/56 ????5’CGACCCAGTGGAAA(C/T)TGATC3′
????39/42 ????5’CGTCC(T/C)AAAGG(A/G)AATTGATC3′
????11/18/44/45 ????5’CGTCCAAGGGGA(A/T)A(C/T)TGATC3′
????16/52 ????5’CGTCCTAAAGGAAACTGATC3′
The downstream inner primer
????6/52/31/39 ????5’TGAAA(A/T)ATAAATTGTAAATCA3′
????33/58 ????5’TGAAAAACAAACTGTA(G/A)(A/G)TCA3′
????44 ????5’TGAAACATAAATTGTAAGTCA3′
????56 ????5’TGAAAAACAAATTGTAATTCA3′
????42 ????5’TGAAATATAAATTGCACATCA3′
????18/45/35/11/16 ????5’TGAAAAATAAACTG(C/T)AAATCA3′
1 of the genotypic general probe of HPV, 15 of HPV genotype specific probes, comprise that HPV genotype HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 etc. amount to 15 kinds of genotype, various probe sequence is as shown in the table
Genotype Sequence is formed
General probe ????5′TATGATTT(A/G)CA(A/G)TTTATG3′
????HPV-6 ????5’TCCGTAACTACATCTTCCA3′
????HPV-11 ????5’TCTGTGTCTAAATCTGCTAC3′
????HPV-16 ????5’CATTATGTGCTGCCATATC3′
????HPV-18 ????5’TGCTTCTACACAGTCTCCT3′
????HPV-31 ????5’GCAATTGCAAACAGTGATAC3′
????HPV-33 ????5’TATGACTTTATGCACACAAGT3′
????HPV-35 ????5’CTGCTGTGTCTTCTAGTGA3′
????HPV-39 ????5’ATAGAGTCTTCCATACCTTC3′
????HPV-42 ????5’TGGTGATACATATACAGCTG3′
????HPV-43 ????5’TCTACTGACCCTACTGTG3′
????HPV-44 ????5’TACTAGTGAACAATATAAGCA3′
????HPV-45 ????5’TACACAAAATCCTGTGCCA3′
????HPV-52 ????5’GAATACCTTCGTCATGGC3′
????HPV-56 ????5’CAGAACAGTTAAGTAAATATG3′
????HPV-58 ????5’ATTATGCACTGAAGTAACTAA3′
Add one section specific polynucleotide tail at the specific probe end, it can be solidificated on the chip, specific polynucleotide tail is 20 the deoxythymidine acid molecules of 3 ' terminal adding at each HPV specific probe.The chip that point is good is placed on and shone in the UV-crosslinked instrument 5 minutes, and probe is completely fixed on the chip, constitute to detect diagnosing chip, the detection diagnosing chip of formation as for depositing under the 2-8 ℃ of condition, as Fig. 1 (A), shown in Figure 2.
Embodiment 2
Probe is set on the chip matrix, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of 5 coordinate point control chips, with the rectangle point sample of coordinate point control chip zone and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, the file of microarray is 3 row, and walking crosswise of microarray is 9 row.Each HPV35, HPV42, HPV43, HPV45, HPV56, the HPV58 probe points is 1, general probe, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV44, the HPV52 probe points is 2, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represents 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, dot matrix is several more than 18, as Fig. 1 (B), shown in Figure 2.
Embodiment 3
Probe is set on the chip matrix, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of 5 coordinate point control chips, with the rectangle point sample of coordinate point control chip zone and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, the file of microarray is 6 row, and walking crosswise of microarray is 5 row.Each HPV35, HPPV42, HPV43, HPV45, HPV56, the HPV58 probe points is 1, general probe, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV44, the HPV52 probe points is 2, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represents 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, dot matrix is several more than 18, as Fig. 1 (C), shown in Figure 2.
Embodiment 4
Probe is set on the chip matrix, comprise 1 of the genotypic general probe of all HV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of 5 coordinate point control chips, with the rectangle point sample of coordinate point control chip zone and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, the file of microarray is 7 row, and walking crosswise of microarray is 4 row.Each HPV35, HPV42, HPV43, HPV45, HPV56, the HPV58 probe points is 1, general probe, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV44, the HPV52 probe points is 2, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represents 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, dot matrix is several more than 18, as Fig. 1 (D), shown in Figure 2.
Embodiment 5
Probe is set on the chip matrix, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of 5 coordinate point control chips, with the rectangle point sample of coordinate point control chip zone and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, the file of microarray is 12 row, and walking crosswise of microarray is more than 3 row.Each HPV35, HPV42, HPV43, HPV45, HPV56, the HPV58 probe points is 1, general probe, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV44, the HPV52 probe points is 2, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represents 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, dot matrix is several more than 18, as Fig. 1 (E), shown in Figure 2.
Embodiment 6
Probe is set on chip matrix, place point sample probe and coordinate mark with microwell plate, probe rectangle microarray on chip is arranged, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of at least 5 above coordinate point control chips, with coordinate point indicate the rectangle point sample zone of control chip and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, more than file at least 3 row of microarray, the walking crosswise more than at least 3 row of microarray; Each HPV35, HPV42, HPV43, HPV45, HPV56, the HPV58 probe points is 1, general probe, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV44, the HPV52 probe points is 2, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represents 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, the dot matrix number is more than at least 18.
The PCR primer adopts the nested primer combination of 2 pairs of 24 chains, and its nucleotide sequence is as shown in the table,
Genotype Sequence is formed
The upstream outer primer
????6/11 ????5’GC(C/T)CAGGGACATAACAATGG3′
????18/58 ????5’GCACA(G/A)GGTCATAACAATGG3′
????39/45 ????5’GCCCAGGGCCA(T/C)AACAATGG3′
????33/35/56 ????5’GCACAAGG(T/C)CATAATAATGG3′
????31/42 ????5’GCTCA(G/A)GGACACAATAATGG3′
????44/52/16 ????5’GC(G/A)CAGGGCCACAATAATGG3′
The upstream inner primer
????6/11/18/31/33 ????5’TTTGTTACTGTGGTAGATAC3′
????44/56 ????5’TTTGTTACTGT(T/A)GTAGATAC3′
????35/58/42 ????5’TTTGTTAC(T/C)GT(A/G)GTTGATAC3′
????52 ????5’TTTGTCACAGTTGTGGATAC3′
????39/45 ????5’TTTGTTACTGT(A/T)GTGGACAC3′
????16 ????5’TTTGTTACTGTTGTTGATAC3′
The downstream outer primer
????6/33/58 ????5’CGTCCCAAAGGA(T/A)ACTGATC3′
????35 ????5’CGGCCCAACGGAAACTGATC3′
????31/56 ????5’CGACCCAGTGGAAA(C/T)TGATC3′
????39/42 ????5’CGTCC(T/C)AAAGG(A/G)AATTGATC
????11/18/44/45 ????5’CGTCCAAGGGGA(A/T)A(C/T)TGATC
????16/52 ????5’CGTCCTAAAGGAAACTGATC3′
The downstream inner primer
????6/52/31/39 ????5’TGAAA(A/T)ATAAATTGTAAATCA3′
????33/58 ????5’TGAAAAACAAACTGTA(G/A)(A/G)TCA3′
????44 ????5’TGAAACATAAATTGTAAGTCA3′
????56 ????5’TGAAAAACAAATTGTAATTCA3′
????42 ????5’TGAAATATAAATTGCACATCA3′
????18/45/35/11/16 ????5’TGAAAAATAAACTG(C/T)AAATCA3′
1 of the genotypic general probe of HPV, 15 of HPV genotype specific probes, comprise that HPV genotype HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amount to 15 kinds of genotype, various probe sequence is as shown in the table
Genotype Sequence is formed
General probe ????5′TATGATTT(A/G)CA(A/G)TTTATG3′
????HPV-6 ????5’TCCGTAACTACATCTTCCA3′
????HPV-11 ????5’TCTGTGTCTAAATCTGCTAC3′
????HPV-16 ????5’CATTATGTGCTGCCATATC3′
????HPV-18 ????5’TGCTTCTACACAGTCTCCT3′
????HPV-31 ????5’GCAATTGCAAACAGTGATAC3′
????HPV-33 ????5’TATGACTTTATGCACACAAGT3′
????HPV-35 ????5’CTGCTGTGTCTTCTAGTGA3′
????HPV-39 ????5’ATAGAGTCTTCCATACCTTC3′
????HPV-42 ????5’TGGTGATACATATACAGCTG3′
????HPV-43 ????5’TCTACTGACCCTACTGTG3′
????HPV-44 ????5’TACTAGTGAACAATATAAGCA3′
????HPV-45 ????5’TACACAAAATCCTGTGCCA3′
????HPV-52 ????5’GAATACCTTCGTCATGGC3′
????HPV-56 ????5’CAGAACAGTTAAGTAAATATG3′
????HPV-58 ????5’ATTATGCACTGAAGTAACTAA3′
Use the dna synthesizer synthesising probing needle, add one section specific polynucleotide tail at the specific probe end, it can be solidificated on the chip, adopt the solid phase phosphoramidite triester method to add 20 deoxythymidine acid molecules at 3 of each HPV specific probe ' end at dna synthesizer.Use the Nunc point sample instrument to carry out point sample at the chip that has positive charge according to pre-set order.
The chip that has positive charge that uses Roche Holding Ag to produce uses the Nunc point sample instrument to carry out point sample by pre-set order, as shown in Figure 1 and Figure 2.
The chip that point is good is placed on the UV-crosslinked instrument internal radiation of UVP and fixes 5 minutes, makes the point sample probe stationary on chip, constitutes and detects diagnosing chip, and is stand-by as for depositing under the 2-8 ℃ of condition the detection diagnosing chip that is made into, as shown in Figure 1 and Figure 2.
Design HPV Auele Specific Primer and probe:, adopt the combination nested primer design of 2 pairs of 24 chains for effectively each genotype HPV of amplification and raising sensitivity.16 of designing probes, comprise 1 of the shared probe of HPV, distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56 and HPV58 and amount to 15 kinds of genotype, as shown in Figure 1 and Figure 2.
Embodiment 7
Take out the chip that obtains hybridization signal, whole chip background is not painted, and hybridization signal is pale brown look, and the difference between specific hybridization point and the non-specific hybridization point is obvious, and by point sample region mode figure, the HPV gene type is found in comparison, as shown in Figure 2.
Interpretation according to clinical samples processing, sleeve type PCR amplification, chip hybridization, chip washing, chip colour developing and chip hybridization signal detects.Carry out clinical samples and handle employing biopsy or paraffin specimen or swab specimen.
The sleeve type PCR amplification is divided into twice to be carried out, and pcr amplification comprises sample disposal supernatant liquor 5 microlitres, a PCR reaction mixture (containing built-up type upstream outer primer or downstream outer primer) 44 microlitres and Taq polysaccharase 1 microlitre for the first time, amounts to 50 microlitre uniform mixing.The PCR cycling condition is 94 ℃ of pre-sex change 2 minutes, 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 35 circulations, keep 72 ℃ to extend 5 minutes.
For the second time pcr amplification is with the first time PCR product 5 microlitres, secondary PCR reaction mixture (containing built-up type upstream inner primer and DIG-dUTP or downstream inner primer and DIG-dUTP) 44 microlitres and Taq polysaccharase 1 microlitre uniform mixing.
The PCR cycling condition is 94 ℃ of pre-sex change 2 minutes, 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 35 circulations, keep 72 ℃ to extend 5 minutes, promptly be made into the hybridization probe of DIG mark through the secondary PCR amplification PCR products.
The chip of making is in advance packed in the hybridization bag, and in hybridization bag, add an amount of hybridization solution, hybridization bag is sealed, carry out 0.5--1 hour prehybridization, add the sex change probe of 5-20 microlitre, put into the hybridization case, carry out 3-6 hour hybridization again, hybridization oven temperature, degree is 45 ℃.
Use 2 * SSC/0.1%SDS washing chip twice at ambient temperature, each 2min, the chip of under 46 ℃ of conditions, washing twice with 0.2 * SSC/0.1%SDS, each 2min uses TNT solution washing chip 5min then.
The chip of washes clean is packed in the clean plastics bag, in plastics bag, add 10% blockade liquid 100 μ l or TN900 μ l, mix, under 37 ℃ condition, place 30min.
Add POD antibody 1 μ l, mix gently, under 37 ℃ condition, place 30min; Chip is taken out usefulness TNT rinsing chip 2 times from plastics bag, each 3min.
Chip is put into clean plastics bag, get DAB colour developing liquid 10 μ l, DAB diluent 10 μ l and aqua sterilisa 480 μ l, be added in the plastics bag immediately after mixing, treat that colour developing reaches (generally developing the color about 5-30 minute) after the desired level, with 1 * PBS termination reaction, obtain the chip hybridization result, whole chip background is not painted, hybridization signal is pale brown look, as shown in Figure 1 and Figure 2.
Embodiment 8
Clinical samples is handled and is adopted biopsy, get and grind after 50 milligrams of biopsies adding dna cleavage liquid 100 microlitres mix, carrying out 55 ℃ of temperature after the grinding bathed 1-3 hour, carrying out 100 ℃ again boiled 10 minutes, with the 12000rpm whizzer centrifugal 10 minutes, get 5 microlitre supernatant liquors and be used as the pcr amplification template, as shown in Figure 1 and Figure 2.
Embodiment 9
Clinical samples is handled and is adopted paraffin specimen, getting 3-5 paraffin specimen section dewaxes, add dna cleavage liquid 50 microlitres, carrying out 55 ℃ of temperature bathed 1-3 hour, carrying out 100 ℃ again boiled 10 minutes, with the whizzer of 12000rpm centrifugal 10 minutes, get 5 microlitre supernatant liquors and be used as the pcr amplification template, as shown in Figure 1 and Figure 2.
Embodiment 10
Clinical samples is handled and is adopted swab specimen, swab specimen soaks with physiological saline, precipitate with centrifugal mode collecting cell, add dna cleavage liquid 50 microlitres, carry out 55 ℃ of temperature and bathed 1-3 hour, carry out 100 ℃ again and boiled 10 minutes, use centrifugal 10 minutes of 12000rpm whizzer, get 5 microlitre supernatant liquors and be used as the pcr amplification template, as shown in Figure 1 and Figure 2.

Claims (6)

1, a kind of diagnosis chip for testing subtype of human papilomavirus gene, it is characterized in that being provided with on the chip matrix probe, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of at least 5 above coordinate point control chips, with the rectangle point sample of coordinate point control chip zone and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, more than the file of microarray at least 3 row, the walking crosswise more than at least 3 row of microarray; Each HPV probe point sample is more than 1, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represent 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, the dot matrix number is more than at least 18; The PCR primer adopts the nested primer combination of 2 pairs of 24 chains, and its nucleotide sequence is as shown in the table,
Genotype Sequence is formed The upstream outer primer ????6/11 ????5’GC(C/T)CAGGGACATAACAATGG?3′ ????18/58 ????5’GCACA(G/A)GGTCATAACAATGG?3′ ????39/45 ????5’GCCCAGGGCCA(T/C)AACAATGG?3′ ????33/35/56 ????5’GCACAAGG(T/C)CATAATAATGG?3′ ????31/42 ????5’GCTCA(G/A)GGACACAATAATGG?3′ ????44/52/16 ????5’GC(G/A)CAGGGCCACAATAATGG?3′ The upstream inner primer ????6/11/18/31/33 ????5’TTTGTTACTGTGGTAGATAC?3′ ????44/56 ????5’TTTGTTACTGT(T/A)GTAGATAC?3′ ????35/58/42 ????5’TTTGTTAC(T/C)GT(A/G)GTTGATAC?3′ ????52 ????5’TTTGTCACAGTTGTGGATAC?3′ ????39/45 ????5’TTTGTTACTGT(A/T)GTGGACAC?3′ ????16 ????5’TTTGTTAC?TGTTGTTGATAC?3′ The downstream outer primer ????6/33/58 ????5’C?GTCCCAAAGGA(T/A)ACTGATC?3′ ????35 ????5’CGGCCCAACGGAAACTGATC?3′ ????31/56 ????5’CGACCCAGTGGAAA(C/T)TGATC?3′ ????39/42 ????5’CGTCC(T/C)AAAGG(A/G)AATTGATC?3′ ????11/18/44/45 ????5’C?GTC?CAAGGGGA(A/T)A(C/T)TGATC?3′ ????16/52 ????5’C?GTC?CTAAAGGAAAC?TGATC?3′ The downstream inner primer ????6/52/31/39 ????5’TGAAA(A/T)ATAAATTGTAAATCA?3′ ????33/58 ????5’TGAAAAACAAACTGTA(G/A)(A/G)TCA?3′ ????44 ????5’TGAAACATAAATTGTAAGTCA?3′ ????56 ????5’TGAAAAACAAATTGTAATTCA?3′ ????42 ????5’TGAAATATAAATTGCACATCA?3′ ????18/45/35/11/16 ????5’TGAAAAATAAACTG(C/T)AAATCA?3′
1 of the genotypic general probe of HPV, 15 of HPV genotype specific probes, comprise that HPV genotype HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amount to 15 kinds of genotype, various probe sequence is as shown in the table
Genotype Sequence is formed General probe ????5′TATGATTT(A/G)CA(A/G)TTTATG?3′ ????HPV-6 ????5’TCCGTAACTACATCTTCCA?3′ ????HPV-11 ????5’TCTGTGTCTAAATCTGCTAC?3′ ????HPV-16 ????5’CATTATGTGCTGCCATATC?3′ ????HPV-18 ????5’TGCTTCTACACAGTCTCCT?3′ ????HPV-31 ????5’GCAATTGCAAACAGTGATAC?3′ ????HPV-33 ????5’TATGACTTTATGCACACAAGT?3′ ????HPV-35 ????5’CTGCTGTGTCTTCTAGTGA?3′ ????HPV-39 ????5’ATAGAGTCTTCCATACCTTC?3′ ????HPV-42 ????5’TGGTGATACATATACAGCTG?3′ ????HPV-43 ????5’TCTACTGACCCTACTGTG?3′ ????HPV-44 ????5’TACTAGTGAACAATATAAGCA?3′ ????HPV-45 ????5’TACACAAAATCCTGTGCCA?3′ ????HPV-52 ????5’GAATACCTTCGTCATGGC?3′ ????HPV-56 ????5’CAGAACAGTTAAGTAAATATG?3′ ????HPV-58 ????5’ATTATGCACTGAAGTAACTAA?3′
Add one section specific polynucleotide tail at the specific probe end, it can be solidificated on the chip, specific polynucleotide tail is to add 20 deoxythymidine acid molecules at 3 of each HPV specific probe ' end; The chip that point is good is placed on and shone in the UV-crosslinked instrument 5 minutes, and probe is completely fixed on the chip, constitutes and detects diagnosing chip, the detection diagnosing chip that constitutes is placed under the 2-8 ℃ of condition deposit.
2, a kind of making method of diagnosis chip for testing subtype of human papilomavirus gene, it is characterized in that on chip matrix, being provided with probe, place point sample probe and coordinate mark with microwell plate, probe rectangle microarray on chip is arranged, comprise 1 of the genotypic general probe of all HPV of identification, 15 of HPV genotype specific probes, be used to distinguish HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amounts to 15 kinds of genotype, probe microarray on chip is arranged, the rectangle point sample zone of at least 5 above coordinate point control chips, with coordinate point indicate the rectangle point sample zone of control chip and point quadrat to, an angle is provided with 2 coordinate point in the microarray, be the point sample starting point, the orientation of indication probe, be 1 coordinate point on 3 angles of other of microarray, more than the file of microarray at least 3 row, the walking crosswise more than at least 3 row of microarray; Each HPV probe repeats 2 of point samples, general probe is the consensus sequence of all HPV, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 represent 15 kinds of genotype, "+" represents positive quality control point, arrangement mode is arranged in order to big number by decimal to various probe by general probe, add feminine gender and positive quality control point, the dot matrix number is more than at least 18; The PCR primer adopts the nested primer combination of 2 pairs of 24 chains, and its nucleotide sequence is as shown in the table,
Genotype Sequence is formed The upstream outer primer ????6/11 ????5’GC(C/T)CAGGGACATAACAATGG?3′ ????18/58 ????5’GCACA(G/A)GGTCATAACAATGG?3′ ????39/45 ????5’GCCCAGGGCCA(T/C)AACAATGG?3′ ????33/35/56 ????5’GCACAAGG(T/C)CATAATAATGG?3′ ????31/42 ????5’GCTCA(G/A)GGACACAATAATGG?3′ ????44/52/16 ????5’GC(G/A)CAGGGCCACAATAATGG?3′ The upstream inner primer ????6/11/18/31/33 ????5’TTTGTTACTGTGGTAGATAC?3′ ????44/56 ????5’TTTGTTACTGT(T/A)GTAGATAC?3′ ????35/58/42 ????5’TTTGTTAC(T/C)GT(A/G)GTTGATAC?3′ ????52 ????5’TTTGTCACAGTTGTGGATAC?3′ ????39/45 ????5’TTTGTTACTGT(A/T)GTGGACAC?3′ ????16 ????5’TTTGTTACTGTTGTTGATAC?3′ The downstream outer primer ????6/33/58 ????5’CGTCCCAAAGGA(T/A)ACTGATC?3′ ????35 ????5’CGGCCCAACGGAAACTGATC?3′ ????31/56 ????5’CGACCCAGTGGAAA(C/T)TGATC?3′ ????39/42 ????5’CGTCC(T/C)AAAGG(A/G)AATTGATC?3′ ????11/18/44/45 ????5’CGTCCAAGGGGA(A/T)A(C/T)TGATC?3′ ????16/52 ????5’CGTCCTAAAGGAAACTGATC?3′ The downstream inner primer ????6/52/31/39 ????5’TGAAA(A/T)ATAAATTGTAAATCA?3′ ????33/58 ????5’TGAAAAACAAACTGTA(G/A)(A/G)TCA?3′ ????44 ????5’TGAAACATAAATTGTAAGTCA?3′ ????56 ????5’TGAAAAACAAATTGTAATTCA?3′ ????42 ????5’TGAAATATAAATTGCACATCA?3′ ????18/45/35/11/16 ????5’TGAAAAATAAACTG(C/T)AAATCA?3′
1 of the genotypic general probe of HPV, 15 of HPV genotype specific probes, comprise that HPV genotype HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV44, HPV45, HPV52, HPV56, HPV58 amount to 15 kinds of genotype, various probe sequence is as shown in the table
Genotype Sequence is formed General probe ????5′TATGATTT(A/G)CA(A/G)TTTATG?3′ ????HPV-6 ????5’TCCGTAACTACATCTTCCA?3′ ????HPV-11 ????5’TCTGTGTCTAAATCTGCTAC?3′ ????HPV-16 ????5’CATTATGTGCTGCCATATC?3′ ????HPV-18 ????5’TGCTTCTACACAGTCTCCT?3′ ????HPV-31 ????5’GCAATTGCAAACAGTGATAC?3′ ????HPV-33 ????5’TATGACTTTATGCACACAAGT?3′ ????HPV-35 ????5’CTGCTGTGTCTTCTAGTGA?3′ ????HPV-39 ????5’ATAGAGTCTTCCATACCTTC?3′ ????HPV-42 ????5’TGGTGATACATATACAGCTG?3′ ????HPV-43 ????5’TCTACTGACCCTACTGTG?3′ ????HPV-44 ????5’TACTAGTGAACAATATAAGCA?3′ ????HPV-45 ????5’TACACAAAATCCTGTGCCA?3′ ????HPV-52 ????5’GAATACCTTCGTCATGGC?3′ ????HPV-56 ????5’CAGAACAGTTAAGTAAATATG?3′ ????HPV-58 ????5’ATTATGCACTGAAGTAACTAA?3′
Use the dna synthesizer synthesising probing needle, add one section specific polynucleotide tail at the specific probe end, it can be solidificated on the chip, adopt the solid phase phosphoramidite triester method to add 20 deoxythymidine acid molecules at 3 of each HPV specific probe ' end at dna synthesizer; Use the Nunc point sample instrument to carry out point sample at the chip that has positive charge according to pre-set order; The chip that point is good is placed on the UV-crosslinked instrument internal radiation of UVP and fixes 5 minutes, makes the point sample probe stationary on chip, constitute to detect diagnosing chip, the detection diagnosing chip that is made into place deposit under the 2-8 ℃ of condition stand-by.
3, a kind of detection method of diagnosis chip for testing subtype of human papilomavirus gene, it is characterized in that taking out the chip that obtains hybridization signal, whole chip background is not painted, hybridization signal is pale brown look, difference between specific hybridization point and the non-specific hybridization point is obvious, by point sample region mode figure, the HPV gene type is found in comparison; Interpretation according to clinical samples processing, sleeve type PCR amplification, chip hybridization, chip washing, chip colour developing and chip hybridization signal detects; Carry out clinical samples and handle employing biopsy or paraffin specimen or swab specimen; The sleeve type PCR amplification is divided into twice to be carried out, and pcr amplification comprises sample disposal supernatant liquor 5 microlitres, a PCR reaction mixture (containing built-up type upstream outer primer or downstream outer primer) 44 microlitres and Taq polysaccharase 1 microlitre for the first time, amounts to 50 microlitre uniform mixing; The PCR cycling condition is 94 ℃ of pre-sex change 2 minutes, 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 35 circulations, keep 72 ℃ to extend 5 minutes; For the second time pcr amplification is with the first time PCR product 5 microlitres, secondary PCR reaction mixture (containing built-up type upstream inner primer and DIG-dUTP or downstream inner primer and DIG-dUTP) 44 microlitres and Taq polysaccharase 1 microlitre uniform mixing; The PCR cycling condition is 94 ℃ of pre-sex change 2 minutes, 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 35 circulations, keep 72 ℃ to extend 5 minutes, promptly be made into the hybridization probe of DIG mark through the secondary PCR amplification PCR products; The chip of making is in advance packed in the hybridization bag, and in hybridization bag, add an amount of hybridization solution, hybridization bag is sealed, carry out 0.5--1 hour prehybridization, add the sex change probe of 5-20 microlitre, put into the hybridization case, carry out 3-6 hour hybridization again, hybridization oven temperature, degree is 45 ℃; Use 2 * SSC/0.1%SDS washing chip twice at ambient temperature, each 2min, the chip of under 46 ℃ of conditions, washing twice with 0.2 * SSC/0.1%SDS, each 2min uses TNT solution washing chip 5min then; The chip of washes clean is packed in the clean plastics bag, in plastics bag, add 10% blockade liquid 100 μ l or TN 900 μ l, mix, under 37 ℃ condition, place 30min; Add POD antibody 1 μ l, mix gently, under 37 ℃ condition, place 30min; Chip is taken out usefulness TNT rinsing chip 2 times from plastics bag, each 3min; Chip is put into clean plastics bag, get DAB colour developing liquid 10 μ l, DAB diluent 10 μ l and aqua sterilisa 480 μ l, be added in the plastics bag immediately after mixing, treat that colour developing reaches (generally developing the color about 5-30 minute) after the desired level, with 1 * PBS termination reaction, obtain the chip hybridization result, whole chip background is not painted, and hybridization signal is pale brown look.
4, the making method of diagnosis chip for testing subtype of human papilomavirus gene according to claim 3, it is characterized in that described clinical samples processing employing biopsy, get and grind after 50 milligrams of biopsies adding dna cleavage liquid 100 microlitres mix, carrying out 55 ℃ of temperature after the grinding bathed 1-3 hour, carrying out 100 ℃ again boiled 10 minutes, with the 12000rpm whizzer centrifugal 10 minutes, get 5 microlitre supernatant liquors and be used as the pcr amplification template.
5, the making method of diagnosis chip for testing subtype of human papilomavirus gene according to claim 3, it is characterized in that described clinical samples processing employing paraffin specimen, getting 3-5 paraffin specimen section dewaxes, add dna cleavage liquid 50 microlitres, carrying out 55 ℃ of temperature bathed 1-3 hour, carry out 100 ℃ again and boiled 10 minutes, centrifugal 10 minutes of the whizzer of usefulness 12000rpm is got 5 microlitre supernatant liquors and is used as the pcr amplification template.
6, the making method of diagnosis chip for testing subtype of human papilomavirus gene according to claim 3, it is characterized in that described clinical samples processing employing swab specimen, swab specimen soaks with physiological saline, precipitate with centrifugal mode collecting cell, add dna cleavage liquid 50 microlitres, carry out 55 ℃ of temperature and bathed 1-3 hour, carry out 100 ℃ again and boiled 10 minutes, with the 12000rpm whizzer centrifugal 10 minutes, get 5 microlitre supernatant liquors and be used as the pcr amplification template.
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