CN104059982A - Oral squarmous cell carcinomas pathogenic gene BPIFB2 and application thereof - Google Patents
Oral squarmous cell carcinomas pathogenic gene BPIFB2 and application thereof Download PDFInfo
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Abstract
The invention relates to an oral squarmous cell carcinomas pathogenic gene BPIFB2 and application of the oral squarmous cell carcinomas pathogenic gene BPIFB2. RNA is extracted from oral squarmous tissue, tissue adjacent to the oral squarmous cell carcinomas and normal tissue, deep sequencing and analyzing are performed on a transcriptome, the oral squarmous cell carcinomas relevant gene BPIFB2 is obtained through screening, an RT-PCR method is adopted for analyzing the expression conditions, on the carcinomas tissue and the tissue adjacent to the carcinomas of a patient with the oral squarmous cell carcinomas, of the screened BPIFB2, and the result shows that the obtained BPIFB2 through screening has good relevance to the oral squarmous cell carcinomas and can be used for preparing an oral squarmous cell carcinomas auxiliary diagnosis preparation or a pregnosis preparation. The invention further discloses a fluorogenic quantitative PCR kit and a using method of the fluorogenic quantitative PCR kit. The kit can detect the expression level of the BPIFB2 in oral squarmous cell carcinomas tissue sensitively, rapidly, quantitatively, accurately and stably and has good application prospects.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly, the present invention relates to transcribe group sequencing analysis by high-throughput and filter out oral carcinoma Disease-causing gene BPIFB2 and described Disease-causing gene in the application of preparing in oral carcinoma auxiliary diagnosis or prognosis preparation.
Background technology
Oral carcinoma (Oral Squarmous Cell Carcinomas, OSCC) is one of common tumour of human body, especially can reach 25% of whole tumours at its sickness rate such as India of developing country, Sri Lanka, Brazil.The sickness rate of oral carcinoma has obvious ascendant trend in developed countries such as America and Europes in recent years, particularly the rejuvenation of age of onset trend.Oral squamous cell carcinomas not only sickness rate is high, and grade malignancy is higher; Often cause voice, chew, swallow, the dysfunction such as face and serious face damage, and threaten patient's life; Five year survival rate only has 60% left and right.Although recently updating and the widespread use of radiotherapy, chemotherapy of oral carcinoma operation method, the five year survival rate of oral carcinoma is not significantly improved.
Cause the major cause of this situation to be: the pathogenesis of oral carcinoma is unclear, Invasion and Metastasis mechanism is not clear, lacks effective early prevention and prognosis judging means.Therefore study the molecular mechanism that oral carcinoma occurs and develops, therefrom finding oral carcinoma early prevention, prognosis judgement and methods for the treatment of is significant problem anxious to be resolved in oral carcinoma research.
Transcription group is type and the copy number of research viable cell contained messenger RNA(mRNA) (Messenger RNA, mRNA) under a certain functional status, has reacted the expression of all genes in a certain specified time and spatial cell.Transcribed group dynamic response the state that grows of cell, under different physiology, pathological state and different cell type all there is the group of transcribing of cell-specific, understand transcribe group be understand molecular function, the moiety of cell and the bioprocess that completes necessary, to we understand life entity body development, disease occurs all have vital role with prevention.
With Roche454, the appearance of the s-generation sequencing technologies that Illumina Solexa and ABI SOLiD are representative, making large-scale application genome and transcription group method solve problem in science becomes possibility.MRNA (the mRNA sequencing that checks order, mRNA-Seq) be the cDNA that mRNA reverse transcription is generated, utilize s-generation high throughput sequencing technologies to carry out cDNA order-checking, obtain rapidly the information of a certain species certain organs or tissue samples nearly all transcript gene expression abundance under a certain state comprehensively.All types of transcripts can adopt same principle high throughput sequencing technologies to carry out degree of depth order-checking, reacts their expression level, is referred to as RNA-Seq.Be accompanied by the commercialization of two generations order-checking platform, especially the application of RNA-Seq technology, up to ten million or even more than one hundred million flux order-checkings have improved the degree of depth and the coverage of transcribing group order-checking greatly, qualitative and quantitative analysis is transcribed and consist of possibility, have promoted the fast development of transcription group.Be widely applied at present biomedical every field and obtained outstanding progress, having made us reach a new level for the molecule of disease and the understanding on genetics basis.These achievements in research are for the cause of disease, the pathogenetic molecular mechanism of parsing disease, special biomarker and the drug target of searching disease of illustrating disease, and then prevention, diagnosis and the treatment level of lifting disease are significant.
What contriver chose by the HiSeq2500 high-flux sequence platform of Illumina company that 8 routine oral carcinoma cancerations, cancer take up healthy tissues transcribes group degree of depth sequencing analysis, preliminary screening goes out to affect oral carcinoma the related gene B PIFB2 developing occurs, and further adopt molecular biology method to confirm the relation of BPIFB2 gene and oral carcinoma: BPIFB2 and oral carcinoma have good dependency, can be used for preparing oral carcinoma auxiliary diagnosis or prognosis preparation, there is important clinical value.
Summary of the invention
The object of the invention has been to provide oral carcinoma pathogenic related gene BPIFB2.
The object of the invention has been to provide a kind of PCR kit for fluorescence quantitative of the BPIFB2 of detection expression level.
Further, the present invention also provides the fluorescence quantification PCR primer of a pair of detection BPIFB2 expression level.
Further, the present invention also provides a kind of PCR kit for fluorescence quantitative using method of the BPIFB2 of detection expression level.
The object of the invention is to provide the application of BPIFB2 gene in diagnosis or the prognosis product of preparing oral carcinoma.
For achieving the above object, three groups of samples that the present invention is taken up healthy tissues to canceration, cancer carry out high-throughput cDNA order-checking, detect and between canceration and cancer beside organism, have 252 difference expression genes, between canceration and healthy tissues, there are 234 difference expression genes, in order better to understand the function of difference expression gene, the function enrichment analysis that contriver carries out Gene Ontology to expressing the gene of imbalance.Finally from difference expression gene, filter out BPIFB2.
The present invention uses oral carcinoma Disease-causing gene BPIFB2 that fluorescence quantifying PCR method Analysis and Screening arrives at the ill patient's of oral carcinoma tumor tissues and the expression of cancer beside organism.
To achieve these goals, the present invention has extracted first respectively the 50 ill patients' of routine oral carcinoma tumor tissues and cancer beside organism, to extracting of its total RNA, 2 primer SEQ ID NO.1 and SEQ ID NO.2 for the BPIFB2 gene that increases are designed, for 2 primers of the reference gene β-actin that increases.Prepare the standard DNA template that contains BPIFB2 gene order, and carried out sensitivity experiments.And then, adopt the method for qRT-PCR to compare the expression level of BPIFB2 gene in oral carcinoma tissue and cancer beside organism.Result shows: qRT-PCR stable amplification result, wherein the expression level of BPIFB2 in cancer beside organism is starkly lower than oral carcinoma tumor tissue and control plasmid 100copies, and BPIFB2 high expression level in oral carcinoma tissue, be that information biology is screened the BPIFB2 gene and the oral carcinoma that obtain and had good dependency, can be used for preparing oral carcinoma auxiliary diagnosis or prognosis preparation.
The object of the invention has been to provide a kind of PCR kit for fluorescence quantitative and using method thereof of the BPIFB2 of detection expression level.This PCR test kit is suitable for existing at present all types fluorescence quantitative gene extender on market, highly sensitive, quantitatively quick and precisely, good stability, has a good application prospect.
The PCR kit for fluorescence quantitative component of a kind of BPIFB2 of detection expression level prepared by the present invention comprises: Auele Specific Primer, internal reference primer, fluorescence quantitative PCR reaction solution.Wherein said Auele Specific Primer comprises upstream primer and downstream primer, and upstream primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.Described internal reference primer is β-actin internal reference primer, and upstream primer sequence is SEQ ID NO.3, and downstream primer sequence is SEQ ID NO.4.Described fluorescence quantitative PCR reaction solution comprises 2 × UltralSYBR One StepRT-qPCR Buffer (With ROX), SuperEnzyme Mix and RNase-Free Water.
The invention also discloses a kind of using method of PCR kit for fluorescence quantitative of the BPIFB2 of detection expression level, quantitative fluorescent PCR system: upstream primer; Downstream primer; Sample RNA10pg-100ng; 2 × UltralSYBROne Step RT-qPCR Buffer (With ROX), SuperEnzyme Mix, adds RNase-Free Water to 25 μ L.Quantitative fluorescent PCR program: 45 DEG C of 10min; 95 DEG C of 5min denaturations, connect 30-40 circulation: 95 DEG C of 10s, 60 DEG C of 45s.
Described test kit also comprises RNA extraction agent.
Described test kit is applied to the detection by quantitative of oral carcinoma BPIFB2 gene, single stage method quantitative fluorescent PCR, and fast and easy, is applicable to clinical detection.
The present invention has also detected this test kit susceptibility, and result shows that this test kit sensing range is 10
6-10
2copies/ μ l, minimum concentrations is 100copies/ μ l.
Advantage of the present invention:
(a) BPIFB2 provided by the invention and oral carcinoma have good dependency, can be used for preparing oral carcinoma auxiliary diagnosis or prognosis preparation.
(b) PCR kit for fluorescence quantitative of detection provided by the invention BPIFB2 expression level has comprised and extracts fluorescent quantitation from RNA and test a whole set of reagent used, and adopt single stage method quantitative fluorescent PCR, both facilitate clinical use, ensured again the consistence of result.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, only for explaining the present invention, and can not be interpreted as limitation of the present invention.Those having ordinary skill in the art will appreciate that: in the situation that not departing from principle of the present invention and aim, can carry out multiple variation, amendment, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.The experimental technique of unreceipted actual conditions in the following example, the condition examinations of conventionally advising according to normal condition or according to manufacturer.
Embodiment 1 studies the collection of sample and the extraction of RNA
Collect 8 routine oral carcinoma and fresh normal tissue, it is divided into groups and is numbered: 3 routine healthy tissues pieces (being numbered N1, N2, N3), 3 routine cancer beside organisms (being numbered P1, P2, P3) piece and 2 routine cancerous tissue pieces (C1 and C2).
Three groups of samples are carried out to RNA extraction, agarose gel electrophoresis after RNA extracts.Tentatively think that from electrophoresis result the RNA sample quality extracting is qualified, can be for further transcribing group analysis.And then by the extraction situation of NanoDrop1000 spectrophotometer detection RNA sample, result is as shown in table 1.Each sample volume is 30 μ l.The sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.RIN (RNAintegrity number) refers to RNA integrity index, is the important indicator of RNA quality.As can be seen from Table 1, the sample of this research all reaches the requirement of transcribing group order-checking.
The Spectrophotometric Assays of table 1 oral carcinoma RNA sample
| Grouping | (ng/ μ l) for concentration | OD(260/280) | (μ l) for volume | (μ g) for total amount | RIN value | Quality type |
| Cancerous tissue | 1274.6 | 2.01 | 30 | 38.2 | 8.7 | Type i |
| Cancer beside organism | 368.4 | 1.94 | 30 | 11.1 | 8.7 | Type i |
| Healthy tissues | 277.2 | 1.99 | 30 | 8.3 | 8.7 | Type II |
Embodiment 2 high-throughputs are transcribed group order-checking and are analyzed
Order-checking platform: the HiSeq2500 high-flux sequence platform of Illumina company
Transcribe group order-checking and coupling:
Canceration, the cancer that we analyze takes up three groups of samples of healthy tissues and comes from 8 routine oral cancer patients.These three groups of samples are carried out to high-throughput cDNA order-checking.From these three tissues, we obtain respectively 4.85 × 107,4.91 × 107, and 4.46 × 107 sections of reading are right.We utilize TopHat that the section of reading is matched to UCSC reference men and women's genome (hg19), the section of reading of unique coupling to scope between 4.04 × 107 to 4.49 × 107, match Ensembl with reference to the section of the reading ratio of gene between 81% to 89%.We check order the average coverage of the degree of depth probably for 130 times of human genome are (according to the total length of the exon region of the unique annotation of Ensembl database, be about 1.13 × 108), only have in addition 1% the section of reading to navigate to rRNA, the database that shows us builds rationally, shows more reliably the expression with the RNA of ployA tail.
The analysis of difference expression gene:
Thereby identify the difference expression gene of different sample rooms in order to calculate the expression of gene, we utilize Cuffdiff method to estimate the expression of gene, thereby the gene of imbalance is expressed in qualification.The standardized expression level of each gene calculates according to the fragment number (FPKM) that matches the long exon region of specific gene 1kb in each 1,000,000 order-checking fragment.FPKM design is decided to be to > 1, takes up healthy tissues respectively in canceration, cancer, we detect 14854-15168 expressing gene, and the mankind that annotate comprising major part are with reference to gene.We further analyze the dependency of genetic expression between different samples.General and the Pearson incidence coefficient height correlation of overall genetic expression.We detect between canceration and cancer beside organism 252 difference expression genes, has 234 difference expression genes between canceration and healthy tissues.
The function enrichment of difference expression gene is analyzed:
In order better to understand the function of difference expression gene, the function enrichment analysis that we carry out GeneOntology to expressing the gene of imbalance.For the Directory of Features of differentiating that cancer is special, the gene that we utilize online tool DAVID to take up to canceration, cancer the expression noticeable change that healthy tissues compares between two carries out function enrichment analysis.We have selected by canceration and cancer, canceration and healthy tissues are expressed the GO catalogue of the significant enrichment of the gene of imbalance in relatively, the difference expression gene between canceration and cancer beside organism is classified as 31 Directories of Features by we, and the difference expression gene between canceration and healthy tissues classifies as 33 Directories of Features.
The selection of candidate gene:
20 candidate genes (normal group vs canceration group, each 10 of the other group of cancer vs canceration group) that screen difference expression gene from above-mentioned RNA-seq order-checking and depth analysis result, sort by is p-value size, selects the BPIFB2 gene ranking the first.
Embodiment 3 oral carcinoma tissues and the BPIFB2 of cancer beside organism expression conditions
One materials and methods
1, material
Oral carcinoma, from 2005-2010 inpatients, is got respectively the 50 ill patients' of routine oral carcinoma tumor tissues and cancer beside organism.
2, method
The extraction of 2.1 oral carcinoma tissues and the total RNA of cancer beside organism
Be that ultrapure RNA of century extracts test kit (Ultrapure RNA Kit (DNase I) by health, article No. CW0597) specification sheets extracts the RNA of oral carcinoma tissue and cancer beside organism, prove the integrity of RNA by gel electrophoresis, measure concentration and the purity of RNA with nucleic acid-protein instrument.
Adopt ultrapure RNA to extract test kit (article No. CW0597) and extract, main operational steps is as follows:
1. sample preparation, 30-50mg adds 1ml Buffer RLT after being organized in liquid nitrogen and fully grinding, or in tissue sample, adds homogenized after 1ml Buffer RLT.
2. after adding Buffer RLT in sample, repeatedly blow and beat several times, make the abundant cracking of sample.Room temperature is placed 5 minutes, and protein nucleic acid mixture is separated completely.
3. add the ratio of 200 μ l chloroforms to add chloroform with every 1ml Buffer RLT, build pipe lid, thermal agitation 15 seconds, room temperature is placed 2 minutes.
4.4 DEG C 12,000rpm (～13,400 × g) centrifugal 10 minutes, now sample is divided into three layers: red organic phase, the colourless water in middle layer and upper strata, RNA mainly, in the water of upper strata, moves on to upper water in a new RNase-Free centrifuge tube (providing for oneself) mutually.
5. in the aqueous phase solution obtaining, add isopyknic 70% ethanol (without the water preparation of RNase), put upside down and mix.
6. upper step gained solution is all joined in the adsorption column (Spin Column RM) that has packed collection tube (Collection Tube2ml) into.If once can not add solution, can proceed to several times.Centrifugal 20 seconds of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column is relay and reclaimed in collector.
7. in adsorption column, add 350 μ l Buffer RW1, centrifugal 20 seconds of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column is relay and reclaimed in collector.
8. preparation DNase I mixed solution: get 52 μ l RNase-Free Water, (1U/ μ l), mixes, and is mixed with the reaction solution that final volume is 80 μ l to add wherein 8 μ l10 × Reaction Buffer and 20 μ l DNase I.
9. in adsorption column, directly add 80 μ l DNase I mixed solutions, hatch 15 minutes for 20-30 DEG C.
10. in adsorption column, add 350 μ l Buffer RW1, centrifugal 1 minute of 10,000rpm, abandons waste liquid, and adsorption column is relay and reclaimed in collector.
11. add 500 μ l Buffer RW2 (whether preoperation inspection has added dehydrated alcohol) in adsorption column, and centrifugal 20 seconds of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column is relay and reclaimed in collector.
12. repeating steps 11.
Centrifugal 2 minutes of 13.12,000rpm, outwells the waste liquid in collection tube.Adsorption column is placed in to room temperature number minute, thoroughly to dry.
14. by adsorption column be placed in one new for RNase centrifuge tube (Collection Tube1.5ml), add 30-50 μ l RNase-Free Water to the middle part of adsorption column, room temperature is placed 1 minute, 12, centrifugal 1 minute of 000rpm, collect RNA solution, preserve RNA for-70 DEG C, prevent degraded.
15. total RNA integrity qualifications: get 2 μ l RNA samples at 1.5% agarose gel electrophoresis (80v, 15min), separate after district's band, FB dyeing, observes Zone electophoresis band under ultraviolet lamp.
16. use nucleic acid-protein instrument are measured concentration and the purity of RNA.
2.2 BPIFB2 detect design of primers with synthetic
According to PCR design of primers principle, the OligoArchitect of application Premier5.0 and enhanced edition
tMsoftware carries out design of primers.
The upstream and downstream primer sequence of BPIFB2 is respectively:
Upstream primer: 5 '-CCAGATCCGCTATTCCAT-3 '; SEQ ID NO.1
Downstream primer: 5 '-AAGAGAACAGCATTGACTTC-3 '; SEQ ID NO.2
Product length is 78bp.
Internal reference β-actin upstream and downstream primer sequence is respectively:
Upstream primer: 5 '-TAATCTTCGCCTTAATACT-3 '; SEQ ID NO.3
Downstream primer: 5 '-CCTTCATACATCTCAAGT-3 '; SEQ ID NO.4
Product length is 103bp.
The foundation of 2.3 quantitative criterion curves
The preparation of standard DNA template
To specifications, from oral carcinoma tissue, utilize ultrapure RNA to extract test kit (article No. CW0597) and extract total RNA, then be that century SuperRT cDNA the first chain synthetic agent box (article No. CW0741) carries out reverse transcription reaction by health, concrete steps are as follows: 1. RNA template, Primer Mix, dNTP Mix, RT Buffer, SuperRT and RNase-Free Water dissolved and be placed in for subsequent use on ice.
2. configuration reaction system, cumulative volume is 20 μ l: final concentration is RNA template, 2 μ lPrimer Mix, 4 μ l dNTP Mix, 4 μ l RT Buffer, the 1 μ l SuperRT of 50pg-5 μ g, adds RNase-Free Water and fills 20 μ l.
3. vortex concussion mixes, of short duration centrifugal, makes the solution on tube wall collect the pipe end.
Hatch 30-50 minute for 4.42 DEG C, hatch 5 minutes for 85 DEG C.After reaction finishes, of short duration centrifugal, be placed in cooled on ice.
The cDNA health that reverse transcription reaction is obtained is that century 2 × Taq MasterMix (article No. CW0682) carries out conventional PCR, and reaction system and condition are as follows: 2 × Taq MasterMix25 μ l, the each 2 μ l of upstream and downstream primer, cDNA0.5 μ g, fill to 50 μ l.Reaction conditions is 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C are extended 30s, 30cycles; Last 72 DEG C are extended 2min.Sample 5 μ L, the product of pcr amplification is carried out to agarose gel electrophoresis detection, cut glue and reclaim and purifying, purified product is connected to pGM-T cloning vector, be transformed into subsequently in DH5 α competent cell.It is the Auele Specific Primer screening positive clone of SEQ ID NO.1 and SEQ ID NO.2 by sequence.After positive colony amplification, extract plasmid DNA, plasmid DNA adopts quantitatively (NanoDropTechnologies of NanoDrop ND-1000 nucleic acid quantification instrument, Wilmington, Delaware) and for the preparation of typical curve, (standard DNA template concentrations scope is 10 as standard substance to do 10 times of serial dilutions
8-10
2copies/ μ l).
2.3 sensitivity experiments
Getting that recombinant plasmid dilutes is in proportion 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2individual copy/μ L, carries out quantitative fluorescent PCR, the detection sensitivity taking the minimum concentration of test positive as the method.The method sensing range that this institute sets up is 10
8-10
2copies/ μ L, minimum concentrations is 100copies/ μ L.
2.4 qRT-PCR detect BPIFB2 gene expression amount
Get the ultrapure RNA of above-mentioned 50 routine oral carcinoma tissues and cancer beside organism and extract test kit (article No. CW0597) and extract total RNA, and then carry out RT-PCR with UltraSYBR single stage method PCR kit for fluorescence quantitative (article No. CW0660).Concrete steps:
1. RNA template, primer, 2 × UltraSYBR One Step RT-qPCR Buffer (With ROX), SuperEnzyme Mi x and RNase-Free Water are dissolved and be placed in for subsequent use on ice.
2.RT-PCR reaction system (25 μ l): 2 × UltraSYBR One Step RT-qPCR Buffer (WithROX), 12.5 μ l, upstream primer (10 μ M) 0.5 μ l, downstream primer (10 μ M) 0.5 μ l, SuperEnzymeMix0.5 μ l, add RNA template (final concentration is 10pg-100ng), RNase-Free Water fills to 25 μ l.
3. vortex concussion mixes, of short duration centrifugal, and solution is collected to the pipe end.
4. thermal cycler is preheating to 45 DEG C, PCR pipe is placed in to thermal cycler, react by following reaction conditions: reverse transcription: 45 DEG C of 10min; 95 DEG C of 5min denaturations, connect 35 circulations: 95 DEG C of 10s, 60 DEG C of 30s.
QRT-PCR reaction result is used to SPSS For Windows11.5 software, and related data adopts χ2-test,chi-square test and the definite stochastic method of Fisher to process, and P < 0.05 has statistical significance; QRT-PCR reaction utilizes MedCalc statistical analysis software to calculate.
Relative quantification formula according to qRT-PCR: 2-Δ Ct × 100%, the relatively expression level of BPIFB2 gene in oral carcinoma tissue and cancer beside organism.Result shows: qRT-PCR stable amplification result, wherein the expression level of BPIFB2 in healthy tissues is between 0.000-0.001, be starkly lower than tumor tissues and control plasmid 100copies, and the expression amount of BPIFB2 in tumor tissues is between 0.002-0.150, these results suggest that BPIFB2 high expression level in oral carcinoma tissue.
4 one kinds of embodiment detect detection kit and the using method of BPIFB2 gene in oral carcinoma
RNA extracts reagent: ultrapure RNA extracts test kit (article No. CW0597)
Fluorescent quantitation reagent: UltraSYBR single stage method PCR kit for fluorescence quantitative (article No. CW0660)
Quantitative fluorescent PCR reaction system and method:
RT-PCR reaction system (25 μ l): 2 × UltraSYBR One Step RT-qPCR Buffer (WithROX), 12.5 μ l, upstream primer (10 μ M) 0.5 μ l, downstream primer (10 μ M) 0.5 μ l, SuperEnzymeMix0.5 μ l, add RNA template (final concentration is 10pg-100ng), RNase-Free Water fills to 25 μ l.
Reaction regulates: reverse transcription: 45 DEG C of 10min; 95 DEG C of 5min denaturations, connect 30-40 circulation: 95 DEG C of 10s, 60 DEG C of 30s.
The present invention adopts the checking of information biology binding molecule biological experiment, filter out oral carcinoma pathogenic related gene BPIFB2, and provide the PCR kit for fluorescence quantitative that detects BPIFB2 expression level, described test kit has comprised and extracts reverse transcription from RNA and test a whole set of reagent used to fluorescent quantitation, both facilitated clinical use, ensure again the consistence of result, there is good potential applicability in clinical practice.
Claims (9)
1. the BPIFB2 gene application in diagnosis or the prognosis product of preparing oral carcinoma.
2. application according to claim 1, is characterized in that, the diagnosis of described oral carcinoma or prognosis product comprise the product that detects BPIFB2 gene in oral carcinoma with RT-PCR, gene chip or immunization method.
3. application according to claim 2, is characterized in that, the primer that the described product for RT-PCR detection oral carcinoma BPIFB2 gene contains a pair of specific amplification BPIFB2 gene; Described gene chip comprises the probe with the nucleic acid array hybridizing of BPIFB2 gene; The described product with immunization method detection oral carcinoma comprises the antibody of being combined with BPIFB2 protein-specific.
4. application according to claim 3, is characterized in that, the primer upstream primer sequence of described a pair of specific amplification BPIFB2 gene is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
5. a PCR kit for fluorescence quantitative that detects oral carcinoma related gene B PIFB2, is characterized in that, described test kit comprises: Auele Specific Primer, internal reference primer, fluorescence quantitative PCR reaction solution.
6. test kit according to claim 5, is characterized in that, described Auele Specific Primer comprises upstream primer and downstream primer, and upstream primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
7. according to the test kit described in claim 5 or 6 any one, it is characterized in that, described test kit also comprises RNA extraction agent.
8. according to the test kit described in claim 5-7 any one, it is characterized in that, quantitative fluorescent PCR system: upstream primer, downstream primer, sample cDNA, 2 × UltralSYBR One Step RT-qPCR Buffer (With ROX), SuperEnzyme Mix, RNase-Free Water; Quantitative fluorescent PCR program: 45 DEG C of 10min; 95 DEG C of 5min denaturations, connect 30-40 circulation: 95 DEG C of 10s, 60 DEG C of 30s.
9. the test kit described in a claim 5-8 any one is in the application of preparing in oral carcinoma auxiliary diagnosis or prognosis preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105891515A (en) * | 2015-11-15 | 2016-08-24 | 陈博 | Kit for specific detection of oral cancer |
| CN105891518A (en) * | 2015-11-15 | 2016-08-24 | 陈博 | Kit for specific detection of oral cancer |
| CN105891487A (en) * | 2015-11-15 | 2016-08-24 | 陈博 | Kit for specific detection of oral cancer |
| CN105891488A (en) * | 2015-11-15 | 2016-08-24 | 陈博 | Kit for specific detection of oral cancer |
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| CN105929163A (en) * | 2015-11-15 | 2016-09-07 | 陈博 | Kit for specific detection of oral cancers |
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| CN105483246A (en) * | 2015-12-29 | 2016-04-13 | 北京泱深生物信息技术有限公司 | Application of differential expression of gene in oral cancer diagnosis |
| CN105483246B (en) * | 2015-12-29 | 2019-01-22 | 北京泱深生物信息技术有限公司 | Application of the differential expression of gene in carcinoma of mouth diagnosis |
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