CN105463083B - A kind of kit and its detection method of the siltation disease gene mutation of detection progressive familial hepatic bile - Google Patents
A kind of kit and its detection method of the siltation disease gene mutation of detection progressive familial hepatic bile Download PDFInfo
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Abstract
The present invention relates to the kit and its detection method of a kind of detection progressive familial hepatic bile siltation disease gene mutation, the kit includes pcr amplification reaction reagent and for ATP8B1 gene/ABCB4 gene/ABCB11 gene magnification PCR primer in sample DNA.Detection method includes sample acquisition, PCR amplification and sequencing.The present invention can be used for detecting ATP8B1 gene/ABCB4 gene/ABCB11 gene mutational site, specific good, high sensitivity；Family member, which carries out screening, can specify onset risk, and Prenatal Screening is simultaneously intervened, and can reduce the disease incidence of offspring's progressive familial hepatic bile siltation disease, application method is simple, has a good application prospect.
Description
Technical field
The invention belongs to kit field, in particular to a kind of detection progressive familial hepatic bile siltation disease gene mutation
Kit and its detection method.
Background technique
Progressive progressive familial intrahepatic cholestasis is a kind of serious cholestatic liver disease, is lost for autosomal recessive
Pass disease.The generation, modification of each functional protein, tune on liver cell and bile duct epithelial cell are mainly caused due to gene mutation
Control defect leads to Hepatocellular cholestasis, and usually in infancy or childhood onset, final development is hepatic failure.According to its cause
Ospc gene is different, and PFIC is broadly divided into 3 types: 1. PFIC1 (Byler disease) is caused by ATP8B1 gene mutation, and the gene is caused to be compiled
P-type ATP enzyme-FIC1 the defect of code, PFIC1 Patients ' Hepatocytes bile acid overload, but how FIC1 defect is in progress into bile
It deposits unclear；2. PFIC2 is derived from the gene of coding cholate drainage pump (bile salt export pump, BSEP) albumen
ABCB11 mutation, affects the expression of bile capillaries film cholate transport protein (BSEP), and cholate secretion is caused to reduce, so that
Cholate is built up and causes seriously to damage in liver cell；3. PFIC3 is the ABCB4 gene for encoding multidrug resistance glycoprotein (MDR3)
Mutation, affects bile capillaries phosphatide transporter, and phosphatide is caused to export obstacle.
The disease is a kind of orphan disease, and definite disease incidence is unknown at present, even if disease incidence is in the U.S., there has been no definite reports
Road, but disease incidence wants higher in some crowds with consanguineous mating cultural custom.This disease men and women's disease incidence is similar, main
Betide infant and children, childhood or adolescence can be lethal because of liver failure.Estimate Neonatal Morbidity about 1/
Between 50000 to 1/100000,10% to the 15% of children's cholestasis reason is accounted for, 10% to the 15% of Lebertransplantationim im Kindesalter is accounted for.
The PFIC clinical manifestation of three kinds of types is different: 1. PFIC1 patient usually just fall ill after birth by several weeks, shows as itch and Huang
Subcutaneous ulcer.Can be in intermittence in morbidity early stage, but gradually developing is persistent symptoms；2. PFIC2 does not have the outer symptom of the liver of PFIC1, and
The clinical manifestation of hepatopathy and disease progression are but violent compared with PFIC1.Some months just shows the cholestatic of duration after birth
Jaundice can progress to acute hepatic failure quickly；3. PFIC3 falls ill a little later, usually in the several leading year of life, minority can be in school age
Even adolescence ability onset, shows as cholestasis and itch, but itch degree is light compared with PFIC1 and PFIC2.All types
PFIC is in worldwide distribution, and 2/3 PFIC case is PFIC1, PFIC2, remaining 1/3 case is PFIC3.
ATP8B1 gene relevant to PFIC1 is located at 18q21-22.Mutation analysis shows that the ATP8B1 gene of PFIC1 is prominent
Becoming mostly is nonsense mutation and deletion mutation, has seriously affected FIC1 protein function, and many FIC1 patients are compound heterozygosis
Son, it is more difficult to shrewd genotype-Phenotype correlation.PFIC2 genotype-Phenotype correlation is not also illustrated, the position ABCB11 (BSEP)
In 2q24-31, contain 28 exons.But most of BSEP are mutated children, no matter its mutation type, liver cell bile capillaries film
Without BSEP protein expression；It has been reported that the mutation of PFIC2 patient BSEP is up to more than 100.Severe phenotype is often and protein truncation
Or the gene mutation of albumen generation failure is related.Wherein it is mostly null mutation, shows as terminator codon in advance appearance and alkali
The insertion or missing of base.And frameshift mutation occurs, remaining is mostly compound missense mutation, these mutation have seriously affected BSEP albumen
Expression and (or) function；It has reported that ABCB4 mutation relevant to PFIC3 is located at 7q21.1 up to more than 30, ABCB4, has contained 28
A exon, overall length 74kb.It is existing researches show that ABCB4 gene mutation hot spot be related to exon 6,9,12,14,18,23,
26, it is respectively positioned on the body region of coding albumen, and the type being mutated is related to the seriousness that gallbladder becomes silted up.Nearly 1/3 case mutation causes to cut
Disconnected albumen generates, and Liver immunity dyeing inspection does not measure MDR3 glycoprotein.Another 2/3 case is missense mutation, mostly occurs and protects in height
That keeps is related to Walker A and the B motif of ATP combination.The variation of these amino acid has no effect on atpase activity and transport process, and
It is to cause intracellular MDR3 glycoprotein assembly defect, functional defect.
PFIC1 type and PFIC2 type clinical phenotypes are similar, by the detection to ATP8B1 and ABCB11 gene, facilitate into
Row is made a definite diagnosis.PFIC is autosomal recessive hereditary diseases, and infant siblings have 25% morbidity, 50% carrying pathogenic mutation
Possibility.Disease-causing gene detection is carried out to asymptomatic siblings and not only helps early detection disease, early treatment.Moreover,
Due to various dcc gene all studied it is clear, so gene diagnosis is most accurate.Sanger PCR sequencing PCR be it is generally acknowledged at present, greatly
The goldstandard of most clinical molecular diagnosis projects, specificity even can reach 100%, be that the classical technology in terms of molecular diagnosis is flat
Platform.Therefore abrupt climatic change is carried out using sequence of the Sanger PCR sequencing PCR to ATP8B1, ABCB11 and ABCB4, there is stability height,
The good advantage of specificity, can effectively avoid the generation of false positive.
In order to provide reliable template to Sanger sequencing it may first have to which the characteristics of being directed to the project establishes a stabilization
Reliable PCR system.In sport technique segment, there are following two difficult points for the part project PCR: 1. projects need to extract blood-based
Because of a group DNA, due to there are numerous uncontrollable factors, often cannot get quality in clinical examination in clinical samples logistics transportation
Excellent whole blood sample, therefore the DNA mass obtained can also have biggish inter-sample difference.2. due to the exon that need to be detected
Number is more, i.e. PCR target fragment is excessive, leads to PCR condition disunity.
Solution temperature (Tm) is a critically important parameter in PCR, is that 50% primer and complementary series show as double-strand
Temperature when DNA molecular, for setting PCR annealing temperature be it is necessary, suitable annealing temperature can effectively reduce non-spy
The opposite sex combines, moreover it is possible to guarantee that aim sequence is effectively annealed.So the Tm value of design primer is as far as possible in the case where guaranteeing specificity
It is close, it is consistent the annealing temperature of PCR, to reach the unification of PCR condition.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of detection progressive familial hepatic bile siltation disease gene is prominent
The kit and its detection method of change, the kit can be used for detecting ATP8B1 gene, ABCB4 gene, ABCB11 gene it is prominent
Conjugate point, specific good, high sensitivity.
A kind of kit of detection progressive familial hepatic bile siltation disease gene mutation of the invention, the kit packet
It includes pcr amplification reaction reagent and the PCR for ATP8B1 gene in sample DNA, ABCB4 gene, ABCB11 gene magnification draws
Object；Wherein, PCR primer is as follows:
ATP8B1 gene:
ATP8B1-4-F:CATAAGCCACCGCACCCAGACA；
ATP8B1-4-R:TCCATTTGCTCCCTTTCCCT；
ATP8B1-5/6-F:TGATGACGGTGATGAGACTTGG；
ATP8B1-5/6-R:GTAATCCCATCTACTCGGAGGCTGA；
ATP8B1-7/8-F:CTTTAATATGTTTACATCCATTAAAGAGC；
ATP8B1-7/8-R:CAGTGGAATGAATGTGCCTT；
ATP8B1-9/10-F:TTTAATTCATTTTTTCCCCATTC；
ATP8B1-9/10-R:TCTTCTTTTGGTTTTGATGGAC；
ATP8B1-11-F:CTGTCTCCTGGGTTCAAGTGATTCT；
ATP8B1-11-R:TCCAGTTCCCGTCATCAAAGCAC；
ATP8B1-12-F:CTAGGAAATGCAAGAGGTTGGAAATC；
ATP8B1-12-R:AAATGAAGGCACTATGTTGGGAGAA；
ATP8B1-13-F:GGGATTCTAACATTCAGACCATAGC；
ATP8B1-13-R:CCAGCAATGCCAGGAGAC；
ATP8B1-14-F:TAGGGCACTGAGGGGCTGAGGA；
ATP8B1-14-R:TGGGCAACCACCAGGAGCAG；
ATP8B1-15-F:ATAATTGAAACCTTGCCTTTGAA；
ATP8B1-15-R:TGTTTCCTACTTTGCTCTTCCAT；
ATP8B1-16-F:TGGCATAACCCTTCCAAGTCAAT；
ATP8B1-16-R:CCCAGCCAACAATACCAGTTTCA；
ATP8B1-17-Fy:AGCCTGGACAACAGAGCAAGAC；
ATP8B1-17-Ry:CAGTTAACATACAGCTTTCATGCAA；
ATP8B1-18-F:AAGTGTATTTGTACAGAATCTGTCATGTT；
ATP8B1-18-R:ACCTTCTTCCATTGTGCCA；
ATP8B1-19-F:GGAGAGCAGCAACCAGGATGT；
ATP8B1-19-R:AAGGAAGAAACTGCTACTGAGGG；
ATP8B1-20-F:TGAATCCTGGAGGTTGAGGCTGT；
ATP8B1-20-R:GCTGAGAAGGAATGGAGAAAAATGG；
ATP8B1-21-F:AAGTCAAAGTTATCTCAGAGTCAAG；
ATP8B1-21-R:CTTTCATGTATAGGCTAAGAGACTT；
ATP8B1-22-F:CAGAATGAAATTCTTCTCGAGA；
ATP8B1-22-R:AAACATCTGCTCTATGAAAACCTA；
ATP8B1-23-F:TGGAACCATTTTTATCAACTGAT；
ATP8B1-23-R:GCACCAGAAACATTTAGAGAAGTC；
ATP8B1-24-F:AGCAAGACCCCCATCTCTATTAAAA；
ATP8B1-24-R:TATTACAAAACAAGAAGCACAGCCA；
ATP8B1-25-F:GCCTTCCTAAAATTTAACAGCAG；
ATP8B1-25-R:TCAGGCAAGAAATAGAAAATGTG；
ATP8B1-26-F:TTGCCTGAGTCAAGGATGGTTTA；
ATP8B1-26-R:AGCCACTGTGCCCAGCCATT；
ATP8B1-27-F:CACCACACCTGGCGAAATATTAA；
ATP8B1-27-R:ATCATTCTTACCAAACTTCCCATGAG；
ATP8B1-28-F:GGGGAGTTCATCAGTGTTCATTTC；
ATP8B1-28-R:ATAGTCCAACCCAAGGAGTTTGTTAT；
ABCB4 gene:
ABCB4-3F:TAAGGCAGGCGACCATTT；
ABCB4-3R:CTCAAGCAACCCTCCCAC；
ABCB4-4F:CTCCTTTTCTAAGACATTCATTT；
ABCB4-4R:AAGATGGTAATGAATAGCAAAAT；
ABCB4-5F:TAAAGAGAAACTTAACAATAGCATC；
ABCB4-5R:ATAGGTGAATCTGGGTAAAGAGTAC；
ABCB4-6-F:GGTATTTAATAGAGCCTTTCTC；
ABCB4-6-R:CCTTGACATATTTTCACACAG；
ABCB4-7F:GTGTTTGTTGGATGTCTACTTCA；
ABCB4-7R:GTGCTGGGATACAGGAATGA；
ABCB4-8F:GAACACTGGCATTTGCTACAT；
ABCB4-8R:GCTTTAGAGTCTGACATTCAACTAT；
ABCB4-9-F:GGTCTCACCATGGGTTCATT；
ABCB4-9-R:TCATCTTTCAAAAAGGAGCGA；
ABCB4-10-F:TAATGAATGCCAGAATGTGAC；
ABCB4-10-R:TCAAAAATATGCAAACTAAAGCCA；
ABCB4-11/12-F:ACTTGTTTGTGCTATGATGGAAT；
ABCB4-11/12-R:AAGGGTGTGAAGGCATTATC；
ABCB4-13-F:GGATGTTTTTCATGAATGGTCC；
ABCB4-13-R:TGGACAATCTTGCATCTCAAA；
ABCB4-14F:CTCAGTTAGGGGTTAAAGGATTA；
ABCB4-14R:CATTTGCTATGTTTCTGTTTCTCA；
ABCB4-15-F:TTTGCCATAATCACGCAGAG；
ABCB4-15-R:TGCTCAGTATAGCATTCACTGGA；
ABCB4-16F:TTGATTGAGAAGCAGTTAGGAAA；
ABCB4-16R:GTATGGCTCATAGTAGCAGTCATCT；
ABCB4-17-F:CGAACAACCCATACTCAGCTTATG；
ABCB4-17-R:GAGGTTGGGAGAAGCAGCAGC；
ABCB4-18F:ATGTGACACTCAAGCCACTATT；
ABCB4-18R:TGAGGGCAAACTTGTAATGT；
ABCB4-19F:GGCAACTGTAAAACAACTCATAACT；
ABCB4-19R:CATCCTTGCGTGAATACCCT；
ABCB4-20F:AGGCTTTGTCTGGTTTTCTTT；
ABCB4-20R:TGGGTATGCTACATGCTTATCTA；
ABCB4-21-F:AGCCAATGTAGCTCAGGCAT；
ABCB4-21-R:AGTTGTAGTGGGCACAAACATT；
ABCB4-22F:TAGTCTTGAACAGATTATGCCTT；
ABCB4-22R:CTTGGAGACCTATTCTTGTTGTT；
ABCB4-23F:AAGGAGGCTGAAGAGATGGT；
ABCB4-23R:AGGATGGAAACTGTGGTAAAT；
ABCB4-24F:TATGAAAATGTATGTCGGGG；
ABCB4-24R:TTAAATGTCAGTCAAGTTGCC；
ABCB4-25F:TTATGTTTCTCTACTACAGTCTTTG；
ABCB4-25R:CAGTTGGGGTTTATAGAATGT；
ABCB4-26F:GCTTAATCACCATAAAAATCATCA；
ABCB4-26R:AGGTAGGCATAATGTATTCCCA；
ABCB4-27F:TACCATTGAGAATTAAAGTCACTAG；
ABCB4-27R:TTGAGGCAAGAGAATCGCT；
ABCB4-28F:ATACAATTTTTGGGATAAGGTGTC；
ABCB4-28R:TGATGACAAACCAGAAACTTATT；
ABCB11 gene:
ABCB11-6-F:GTAATCTCTGGTGGCTTGATCCTA；
ABCB11-6-R:TTGTATCCTCCTAAGTTTCCATCTG；
ABCB11-7-F:AATTACTTTCCCCCTTTTCTCAA；
ABCB11-7-R:AACATAGGAAAACTGAAAATATTTTTAAAT；
ABCB11-8-F:TAGGGATAGAGAGATGGGAATGT；
ABCB11-8-R:GGGAGTAATCTAACAAACTACATGA；
ABCB11-9-F:CAGACTGACTTACCTAATTTCTTGGACT；
ABCB11-9-R:AAACATACTGCTAAAGGCTTGGGACT；
ABCB11-10-F:TCCCTGAAGCTGCTCTGTGTTTG；
ABCB11-10-R:GAAGGAAATGCTATGTCTCGGTC；
ABCB11-11-F:GCTACCTTGCTTAGACTTCTTACT；
ABCB11-11-R:TTCTTCAGGAGTTCATTCTGTGCC；
ABCB11-12-F:CTAAGAGGCACAATAAATGTATACATCTTCA；
ABCB11-12-R:GAAACAGAGTCAGGCTTCAGAAAAT；
ABCB11-13-F:CTCATCCTTGCCAATGTTTCCT；
ABCB11-13-R:AAGCGTGTCCCATCAATTCAGTA；
ABCB11-14-F:AGAATCTTATTGGCCTCTATTTTTTCTGC；
ABCB11-14-R:TTGGGAATCATACGAGAAGAAATGTGTA；
ABCB11-15-F:GACAGAAAGGACATTATAGTGG；
ABCB11-15-R:AATCAGACTAGATGCATGAACCC；
ABCB11-16-F:GATGCAAAGGTCAGTGTCAGCT；
ABCB11-16-R:TGCCAGAGTTGTTGGGAGAA；
ABCB11-17-F:GGTTTAAATGACTCAGTCTTGC；
ABCB11-17-R:TGAGGATTAGGACTACAGAGGA；
ABCB11-18-F:TACCATGTGACCTACCAAACATTTCTA；
ABCB11-18-R:CCTAGAAAACTCATATTCTCAGGCTTAA；
ABCB11-19-F:TGTGAATGCCAAAGGATCTGC；
ABCB11-19-R:TTGCCTTCTTACCCTCTGTGTG；
ABCB11-20/21-F:CCACAGCTTACATTAGGGTTCACTC；
ABCB11-20/21-R:GAATGCTCTAATGAAAGAATGCC；
ABCB11-22-F:ATATTTTATCACAACTTTATAAAAGGTCTGA；
ABCB11-22-R:GCTTCCTTCAGTCTCTTCGTACTACT；
ABCB11-23-F:GCAGCCACTGAAATGTCACGAA；
ABCB11-23-R:ACCAGGCTATTCCTTCCTTGTGT；
ABCB11-24-F:ATATTTGGTCCTTTCCTGGCAGAAC；
ABCB11-24-R:TACCCCACACCATCCCCTGA；
ABCB11-25-F:TTTGGCAGCATGGTTTGAAGG；
ABCB11-25-R:GAGTCTGGCAAAGCAAAAACTTGAAG；
ABCB11-26-F:ATGGATGCCACTCCTGATAGACA；
ABCB11-26-R:GGATTTTCAGATTAGGGATGCTC；
ABCB11-27-F:TCACTCACTGTTCCCTAGTTCAA；
ABCB11-27-R:GCTCTAGATAATTGTCTTTTGG；
ABCB11-28-F:CATCAACTTTCCATCTTCTCTTTGC；
ABCB11-28-R:TTGGGTTTTCCCTCATATGGAC.
The pcr amplification reaction reagent includesArchaeal dna polymerase, dNTPs, MgCl2And reaction buffer.
A kind of detection method of the kit of detection progressive familial hepatic bile siltation disease gene mutation of the invention, packet
Include following steps:
(1) blood sample to be measured is acquired, DNA is extracted；
(2) using the DNA as template, using for ATP8B1 gene, ABCB4 gene, ABCB11 gene magnification in sample DNA
PCR primer carry out PCR amplification, obtain PCR reaction product, carry out sequencing analysis, it is determined whether there are base mutations.
PCR reaction condition in the step (2) are as follows: 95 DEG C of 5min；95 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 40s, 15 are followed
Ring；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, 25 circulations；Last 72 DEG C of 10min.
PCR reaction system in the step (2) are as follows: pcr amplification reaction reagent 10 μ L, ddH26 μ L of O, upstream and downstream primer
Each 1 μ L, 2 μ L of sample DNA.
Beneficial effect
The present invention can be used for detecting the mutational site of ATP8B1 gene, ABCB4 gene, ABCB11 gene, and specificity is good, spirit
Sensitivity is high；It can be used for the clinical complementary finger clarified a diagnosis as progressive familial hepatic bile siltation disease early detection, in early days
Mark reduces misdiagnosis rate and the treatment that avoids delay；Family member, which carries out screening, can specify onset risk, and Prenatal Screening is simultaneously done
In advance, the disease incidence of offspring's progressive familial hepatic bile siltation disease can be reduced, application method is simple, before having good application
Scape.
Detailed description of the invention
Fig. 1 is the sequencing peak figure of ATP8B1 (A), ABCB4 (B) and ABCB11 (C) genic mutation type.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
1. sample extracts
(1) whole blood for extracting 200 μ L, is added 20 μ L Proteinase K Solutions, mixes.
(2) 200 μ L buffer GB are added, are sufficiently mixed by inversion, 56 DEG C are placed 10 minutes, are mixed by inversion therebetween for several times, molten
Liquid strain is limpid, and brief centrifugation is to remove the droplet of cap wall.
(3) 200 μ L dehydrated alcohols are added, are sufficiently mixed by inversion, at this time it is possible that flocculent deposit, brief centrifugation is to go
Except the droplet of cap wall.
(4) previous step acquired solution and flocculent deposit be all added in an adsorption column CB3 (adsorption column is put into collecting pipe
In), 12000rpm (13400 × g) is centrifuged 30 seconds, outwells the waste liquid in collecting pipe, adsorption column CB3 is put back in collecting pipe.
(5) 500 μ L buffer GD (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12000rpm (13400 × g) is centrifuged 30 seconds, outwells waste liquid, adsorption column CB3 is put into collecting pipe.
(6) 600 μ L rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12000rpm (13400 × g) is centrifuged 30 seconds, outwells waste liquid, adsorption column CB3 is put into collecting pipe.
(7) 600 μ L rinsing liquid PW, 12000rpm (13400 × g) are added into adsorption column CB3 to be centrifuged 30 seconds, outwell useless
Liquid.
(8) adsorption column CB3 is put back in collecting pipe, 12000rpm (13400 × g) is centrifuged 2 minutes, outwells waste liquid.It will inhale
Attached column CB3, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material.
(9) adsorption column CB3 is transferred in a clean 1.5mL centrifuge tube, is vacantly added dropwise to the intermediate position of adsorbed film
50 μ L elution buffer TE, are placed at room temperature for 2-5 minutes, and 12000rpm (13400 × g) is centrifuged 2 minutes, and solution is collected into centrifugation
Guan Zhong.
2. experimentation
(1) pre-mixed PCR is taken to react by sample number n (sample number=1+positive control of number of awaiting test sample+negative control 1)
The every 20 μ L of pipe of system is sub-packed in reaction tube.
(2) it respectively takes 2 μ L to be separately added into reaction tube the above-mentioned sample to be tested handled well and negative, positive control, mixes,
The low-speed centrifugal several seconds carries out PCR amplification, and specific reaction system is as follows:
PCR reaction condition, see the table below:
3. sequencing analysis
ATP8B1 sequencing primer sequence
ABCB4 sequencing primer sequence
ABCB11 sequencing primer sequence
4. specific step is as follows for Sanger sequencing:
4.1 sequencing PCR:
4.1.1 preparation of reagents: according to the form below carries out, and operates on ice, -20 DEG C of refrigerators are stored in after packing.
BigDye system:
Enzyme purification system:
| Raw material | 1X | Need volume (ul/ pipe) |
| CIP | 0.1ul | - |
| Exo I | 0.5ul | - |
| Deionized water | 1.4ul | - |
The purifying of 4.2 PCR products
4.2.1 the purifying reaction solution dispensed is taken out, in the good corresponding number of tube wall subscript.
4.2.2 it takes 9ulPCR product to be added in the pipe accordingly numbered, mixes (attention cannot generate bubble).
4.2.3 sample will be mixed according to PCR instrument on following reaction condition, carry out enzyme purification amplification.
37℃→95℃→4℃
50′ 5′ +∞
4.2.4 purified sample is used for next step sequencing reaction, as the same day does not have to put freezen protective.
4.3 sequencing reaction
4.3.1 each sample does positive and negative two reactions, and label is finished writing on corresponding thin-wall tube.
4.3.2 every reaction system 5ul, BigDye mixed liquor and 5P primer are fixed plus 1ul, template generally add 2ul, maximum
For 3ul (adjusting according to PCR product electrophoresis result), 5ul is supplied with water.
4.3.3 the water for first adding corresponding amount, is then added corresponding 1ul primer and is added in water, then in the different places of tube wall
The BigDye mixed liquor for adding 1ul, is eventually adding template.
4.3.4 the of short duration centrifugation of lid upper tube cap low speed, vortice mix, the of short duration centrifugation of low speed again.
4.3.5 according to PCR instrument on following reaction condition, sequencing reaction is carried out.
95℃→95℃→56℃→60℃→4℃
4′ 15” 20” 2′ +∞
25 circulations.
4.4. ethanol purification after sequencing reaction, upper machine.
4.4.1 the PCR product expanded is subjected to of short duration centrifugation.Every pipe side it is adherent be added 2ul 125mM EDTA,
Then it in the adherent addition 15ul dehydrated alcohol in the other side of pipe, is vortexed and mixes.
4.4.2 with board-like centrifuge, 4000rpm, centrifugation 30min, then 300rpm is inverted brief centrifugation, removes supernatant.
4.4.3 20 DEG C of 75% ethyl alcohol of pre-cooling of 50ul are added, is vortexed and mixes, 4000rpm, 4 DEG C of centrifugation 15min.
4.4.4 300rpm is inverted brief centrifugation, removes supernatant, is then placed into room temperature, 15min is evaporated completely ethyl alcohol.
4.4.5 after ethyl alcohol volatilizees completely, water and Hi-Di is added in every pipeTMEach 5ul, sufficient vortex vibrate 1min., it is of short duration from
15min. is stood after the heart is completely dissolved sequencing product.
4.4.6 by Hi-DiTMLysate is transferred in the corresponding position of 96 orifice plates.It puts to PCR amplification instrument, 95 DEG C pre-
It is denaturalized 5min., is transferred quickly to 4 DEG C of cooling 5min on ice chest.Machine is sequenced and analyzes on to 3500 sequenators.
Data analysis: sequence alignment analysis is carried out using sequencing analysis software.The sequence that sequencing is obtained
It is compared with the standard sequence retrieved in NCBI, the gene order of confirmatory sample.
5. clinical samples compare: preclinical Samples detection 30, kit test result coincidence rate 90%.
Therefore, using kit of the present invention, using sequencing technologies, to ATP8B1 gene, ABCB4 gene, ABCB11 gene
Mutation is detected.It can be detected and progressive familial hepatic bile by specific primer and with the ddNTP of fluorescent marker
Deposit the relevant gene mutation site of disease.It can be used for early detection, the early stage of clinical progressive familial hepatic bile siltation disease
The early screening method of Subsidiary Index and progressive familial hepatic bile the siltation disease patient to clarify a diagnosis.
Claims (2)
1. a kind of kit of detection progressive familial hepatic bile siltation disease gene mutation, it is characterised in that: the kit
Including pcr amplification reaction reagent and for ATP8B1 gene in sample DNA, ABCB4 gene, ABCB11 gene magnification PCR
Primer；Wherein, PCR primer is as follows:
ATP8B1 gene:
ATP8B1-4-F:CATAAGCCACCGCACCCAGACA；
ATP8B1-4-R:TCCATTTGCTCCCTTTCCCT；
ATP8B1-5/6-F:TGATGACGGTGATGAGACTTGG；
ATP8B1-5/6-R:GTAATCCCATCTACTCGGAGGCTGA；
ATP8B1-7/8-F:CTTTAATATGTTTACATCCATTAAAGAGC；
ATP8B1-7/8-R:CAGTGGAATGAATGTGCCTT；
ATP8B1-9/10-F:TTTAATTCATTTTTTCCCCATTC；
ATP8B1-9/10-R:TCTTCTTTTGGTTTTGATGGAC；
ATP8B1-11-F:CTGTCTCCTGGGTTCAAGTGATTCT；
ATP8B1-11-R:TCCAGTTCCCGTCATCAAAGCAC；
ATP8B1-12-F:CTAGGAAATGCAAGAGGTTGGAAATC；
ATP8B1-12-R:AAATGAAGGCACTATGTTGGGAGAA；
ATP8B1-13-F:GGGATTCTAACATTCAGACCATAGC；
ATP8B1-13-R:CCAGCAATGCCAGGAGAC；
ATP8B1-14-F:TAGGGCACTGAGGGGCTGAGGA；
ATP8B1-14-R:TGGGCAACCACCAGGAGCAG；
ATP8B1-15-F:ATAATTGAAACCTTGCCTTTGAA；
ATP8B1-15-R:TGTTTCCTACTTTGCTCTTCCAT；
ATP8B1-16-F:TGGCATAACCCTTCCAAGTCAAT；
ATP8B1-16-R:CCCAGCCAACAATACCAGTTTCA；
ATP8B1-17-Fy:AGCCTGGACAACAGAGCAAGAC；
ATP8B1-17-Ry:CAGTTAACATACAGCTTTCATGCAA；
ATP8B1-18-F:AAGTGTATTTGTACAGAATCTGTCATGTT；
ATP8B1-18-R:ACCTTCTTCCATTGTGCCA；
ATP8B1-19-F:GGAGAGCAGCAACCAGGATGT；
ATP8B1-19-R:AAGGAAGAAACTGCTACTGAGGG；
ATP8B1-20-F:TGAATCCTGGAGGTTGAGGCTGT；
ATP8B1-20-R:GCTGAGAAGGAATGGAGAAAAATGG；
ATP8B1-21-F:AAGTCAAAGTTATCTCAGAGTCAAG；
ATP8B1-21-R:CTTTCATGTATAGGCTAAGAGACTT；
ATP8B1-22-F:CAGAATGAAATTCTTCTCGAGA；
ATP8B1-22-R:AAACATCTGCTCTATGAAAACCTA；
ATP8B1-23-F:TGGAACCATTTTTATCAACTGAT；
ATP8B1-23-R:GCACCAGAAACATTTAGAGAAGTC；
ATP8B1-24-F:AGCAAGACCCCCATCTCTATTAAAA；
ATP8B1-24-R:TATTACAAAACAAGAAGCACAGCCA；
ATP8B1-25-F:GCCTTCCTAAAATTTAACAGCAG；
ATP8B1-25-R:TCAGGCAAGAAATAGAAAATGTG；
ATP8B1-26-F:TTGCCTGAGTCAAGGATGGTTTA；
ATP8B1-26-R:AGCCACTGTGCCCAGCCATT；
ATP8B1-27-F:CACCACACCTGGCGAAATATTAA；
ATP8B1-27-R:ATCATTCTTACCAAACTTCCCATGAG；
ATP8B1-28-F:GGGGAGTTCATCAGTGTTCATTTC；
ATP8B1-28-R:ATAGTCCAACCCAAGGAGTTTGTTAT；
ABCB4 gene:
ABCB4-3F:TAAGGCAGGCGACCATTT；
ABCB4-3R:CTCAAGCAACCCTCCCAC；
ABCB4-4F:CTCCTTTTCTAAGACATTCATTT；
ABCB4-4R:AAGATGGTAATGAATAGCAAAAT；
ABCB4-5F:TAAAGAGAAACTTAACAATAGCATC；
ABCB4-5R:ATAGGTGAATCTGGGTAAAGAGTAC；
ABCB4-6-F:GGTATTTAATAGAGCCTTTCTC；
ABCB4-6-R:CCTTGACATATTTTCACACAG；
ABCB4-7F:GTGTTTGTTGGATGTCTACTTCA；
ABCB4-7R:GTGCTGGGATACAGGAATGA；
ABCB4-8F:GAACACTGGCATTTGCTACAT；
ABCB4-8R:GCTTTAGAGTCTGACATTCAACTAT；
ABCB4-9-F:GGTCTCACCATGGGTTCATT；
ABCB4-9-R:TCATCTTTCAAAAAGGAGCGA；
ABCB4-10-F:TAATGAATGCCAGAATGTGAC；
ABCB4-10-R:TCAAAAATATGCAAACTAAAGCCA；
ABCB4-11/12-F:ACTTGTTTGTGCTATGATGGAAT；
ABCB4-11/12-R:AAGGGTGTGAAGGCATTATC；
ABCB4-13-F:GGATGTTTTTCATGAATGGTCC；
ABCB4-13-R:TGGACAATCTTGCATCTCAAA；
ABCB4-14F:CTCAGTTAGGGGTTAAAGGATTA；
ABCB4-14R:CATTTGCTATGTTTCTGTTTCTCA；
ABCB4-15-F:TTTGCCATAATCACGCAGAG；
ABCB4-15-R:TGCTCAGTATAGCATTCACTGGA；
ABCB4-16F:TTGATTGAGAAGCAGTTAGGAAA；
ABCB4-16R:GTATGGCTCATAGTAGCAGTCATCT；
ABCB4-17-F:CGAACAACCCATACTCAGCTTATG；
ABCB4-17-R:GAGGTTGGGAGAAGCAGCAGC；
ABCB4-18F:ATGTGACACTCAAGCCACTATT；
ABCB4-18R:TGAGGGCAAACTTGTAATGT；
ABCB4-19F:GGCAACTGTAAAACAACTCATAACT；
ABCB4-19R:CATCCTTGCGTGAATACCCT；
ABCB4-20F:AGGCTTTGTCTGGTTTTCTTT；
ABCB4-20R:TGGGTATGCTACATGCTTATCTA；
ABCB4-21-F:AGCCAATGTAGCTCAGGCAT；
ABCB4-21-R:AGTTGTAGTGGGCACAAACATT；
ABCB4-22F:TAGTCTTGAACAGATTATGCCTT；
ABCB4-22R:CTTGGAGACCTATTCTTGTTGTT；
ABCB4-23F:AAGGAGGCTGAAGAGATGGT；
ABCB4-23R:AGGATGGAAACTGTGGTAAAT；
ABCB4-24F:TATGAAAATGTATGTCGGGG；
ABCB4-24R:TTAAATGTCAGTCAAGTTGCC；
ABCB4-25F:TTATGTTTCTCTACTACAGTCTTTG；
ABCB4-25R:CAGTTGGGGTTTATAGAATGT；
ABCB4-26F:GCTTAATCACCATAAAAATCATCA；
ABCB4-26R:AGGTAGGCATAATGTATTCCCA；
ABCB4-27F:TACCATTGAGAATTAAAGTCACTAG；
ABCB4-27R:TTGAGGCAAGAGAATCGCT；
ABCB4-28F:ATACAATTTTTGGGATAAGGTGTC；
ABCB4-28R:TGATGACAAACCAGAAACTTATT；
ABCB11 gene:
ABCB11-6-F:GTAATCTCTGGTGGCTTGATCCTA；
ABCB11-6-R:TTGTATCCTCCTAAGTTTCCATCTG；
ABCB11-7-F:AATTACTTTCCCCCTTTTCTCAA；
ABCB11-7-R:AACATAGGAAAACTGAAAATATTTTTAAAT；
ABCB11-8-F:TAGGGATAGAGAGATGGGAATGT；
ABCB11-8-R:GGGAGTAATCTAACAAACTACATGA；
ABCB11-9-F:CAGACTGACTTACCTAATTTCTTGGACT；
ABCB11-9-R:AAACATACTGCTAAAGGCTTGGGACT；
ABCB11-10-F:TCCCTGAAGCTGCTCTGTGTTTG；
ABCB11-10-R:GAAGGAAATGCTATGTCTCGGTC；
ABCB11-11-F:GCTACCTTGCTTAGACTTCTTACT；
ABCB11-11-R:TTCTTCAGGAGTTCATTCTGTGCC；
ABCB11-12-F:CTAAGAGGCACAATAAATGTATACATCTTCA；
ABCB11-12-R:GAAACAGAGTCAGGCTTCAGAAAAT；
ABCB11-13-F:CTCATCCTTGCCAATGTTTCCT；
ABCB11-13-R:AAGCGTGTCCCATCAATTCAGTA；
ABCB11-14-F:AGAATCTTATTGGCCTCTATTTTTTCTGC；
ABCB11-14-R:TTGGGAATCATACGAGAAGAAATGTGTA；
ABCB11-15-F:GACAGAAAGGACATTATAGTGG；
ABCB11-15-R:AATCAGACTAGATGCATGAACCC；
ABCB11-16-F:GATGCAAAGGTCAGTGTCAGCT；
ABCB11-16-R:TGCCAGAGTTGTTGGGAGAA；
ABCB11-17-F:GGTTTAAATGACTCAGTCTTGC；
ABCB11-17-R:TGAGGATTAGGACTACAGAGGA；
ABCB11-18-F:TACCATGTGACCTACCAAACATTTCTA；
ABCB11-18-R:CCTAGAAAACTCATATTCTCAGGCTTAA；
ABCB11-19-F:TGTGAATGCCAAAGGATCTGC；
ABCB11-19-R:TTGCCTTCTTACCCTCTGTGTG；
ABCB11-20/21-F:CCACAGCTTACATTAGGGTTCACTC；
ABCB11-20/21-R:GAATGCTCTAATGAAAGAATGCC；
ABCB11-22-F:ATATTTTATCACAACTTTATAAAAGGTCTGA；
ABCB11-22-R:GCTTCCTTCAGTCTCTTCGTACTACT；
ABCB11-23-F:GCAGCCACTGAAATGTCACGAA；
ABCB11-23-R:ACCAGGCTATTCCTTCCTTGTGT；
ABCB11-24-F:ATATTTGGTCCTTTCCTGGCAGAAC；
ABCB11-24-R:TACCCCACACCATCCCCTGA；
ABCB11-25-F:TTTGGCAGCATGGTTTGAAGG；
ABCB11-25-R:GAGTCTGGCAAAGCAAAAACTTGAAG；
ABCB11-26-F:ATGGATGCCACTCCTGATAGACA；
ABCB11-26-R:GGATTTTCAGATTAGGGATGCTC；
ABCB11-27-F:TCACTCACTGTTCCCTAGTTCAA；
ABCB11-27-R:GCTCTAGATAATTGTCTTTTGG；
ABCB11-28-F:CATCAACTTTCCATCTTCTCTTTGC；
ABCB11-28-R:TTGGGTTTTCCCTCATATGGAC.
2. a kind of kit of detection progressive familial hepatic bile siltation disease gene mutation according to claim 1,
Be characterized in that: the pcr amplification reaction reagent includesArchaeal dna polymerase, dNTPs, MgCl2And reaction buffer.
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