CN105463083A - Kit for detecting gene mutation of progressive familial intrahepatic choleatasia (PFIC) and detection method of kit - Google Patents
Kit for detecting gene mutation of progressive familial intrahepatic choleatasia (PFIC) and detection method of kit Download PDFInfo
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Abstract
The invention relates to a kit for detecting gene mutation of progressive familial intrahepatic choleatasia (PFIC) and a detection method of the kit. The kit comprises a PCR amplification reaction agent and a PCR primer for amplifying ATP8B1 gene/ABCB4 gene/ABCB11 gene in sample DNA. The detection method comprises the steps of sample collection, PCR amplification and sequencing. The kit and the detection method can be used for detecting the mutation sites of ATP8B1 gene/ABCB4 gene/ABCB11 gene, and are good in specificity and high in sensitivity; after family members are screened, the risk of disease is clear; prenatal screening and intervention are carried out, so that the incidence of PFIC of the descendants is reduced; a method of the kit is simple; and the kit and the detection method have good application prospects.
Description
Technical field
The invention belongs to test kit field, particularly a kind of test kit and detection method thereof detecting the transgenation of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease.
Background technology
Progressive familial intrahepatic cholestasis disease is a kind of serious cholestatic liver disease, is autosomal recessive disease.Mainly cause due to transgenation the generation of each functional protein on liver cell and bile duct epithelial cell, modification, regulation and control defect to cause Hepatocellular cholestasis, usually at infancy or childhood onset, finally develop into liver failure.Different according to its Disease-causing gene, PFIC is mainly divided into 3 types: 1. PFIC1 (Byler is sick) is caused by ATP8B1 transgenation, cause the P type ATP enzyme-FIC1 defect of this genes encoding, the overload of PFIC1 Patients ' Hepatocytes bile acide, but how FIC1 defect is in progress into cholestasis it be unclear that; 2. PFIC2 comes from coding cholate drainage pump (bilesaltexportpump, BSEP) the Gene A BCB11 sudden change of albumen, have impact on the expression of bile capillary film cholate translocator (BSEP), cause cholate to be secreted to reduce, thus make cholate in liver cell build up and cause major injury; 3. PFIC3 is the sudden change of the ABCB4 gene of coding multidrug resistance glycoprotein (MDR3), have impact on bile capillary phosphatide transporter, causes phosphatide to export obstacle.
This disease is a kind of orphan disease, and its definite sickness rate is failed to understand at present, even if sickness rate not yet has definite report in the U.S., but it is higher to have sickness rate in the crowd of consanguineous mating cultural custom at some.This sick men and women's sickness rate is similar, mainly betides infant and children, Childhood or pubescence can be lethal because of liver failure.Estimate, between Neonatal Morbidity about 1/50000 to 1/100000, to account for 10% to 15% of children's cholestasis reason, account for 10% to 15% of Lebertransplantationim im Kindesalter.The PFIC clinical manifestation of three kinds of types is different: 1. PFIC1 patient's just morbidity of a few week after birth usually, shows as itch and jaundice.In intermittent, but persistent symptoms can be developed into gradually in early days in morbidity; 2. PFIC2 does not have the outer symptom of the liver of PFIC1, and the clinical manifestation of hepatopathy and disease progression comparatively PFIC1 is violent.After birth, some months just shows the obstructive jaundice of persistence, can be in progress very soon as acute hepatic failure; 3. PFIC3 morbidity is a little later, and usually in the several leading year of life, minority in school age even adolescence just onset, can show as cholestasis and itch, but itch degree comparatively PFIC1 and PFIC2 is light.All types PFIC is all in worldwide distribution, and the PFIC case of 2/3 is PFIC1, PFIC2, and all the other 1/3 cases are PFIC3.
The ATP8B1 gene relevant to PFIC1 is positioned at 18q21-22.Mutation analysis shows, mostly the ATP8B1 transgenation of PFIC1 is nonsense mutation and deletion mutantion, has had a strong impact on FIC1 protein function, and many FIC1 patients are compound heterozygotes, is more difficult to shrewd genotype-Phenotype dependency.PFIC2 genotype-Phenotype dependency does not also understand, ABCB11 (BSEP) is positioned at 2q24-31, containing 28 exons.The children but most of BSEP suddenlys change, no matter its mutation type, liver cell bile capillary film is all without BSEP protein expression; Now report that the sudden change of PFIC2 patient B SEP can reach more than 100 and plant.Severe phenotype often generates exhaustion transgenation with protein truncation or albumen is relevant.Wherein be mostly null mutation, show as insertion or the disappearance of terminator codon in advance appearance and base.And there is phase shift mutation, more than mostly be compound missense mutation, these sudden changes have all had a strong impact on expression and (or) the function of BSEP albumen; Reported that the ABCB4 sudden change relevant to PFIC3 reaches more than 30 and plant, ABCB4 is positioned at 7q21.1, containing 28 exons, and total length 74kb.Existing research display ABCB4 transgenation focus relates to exon 6,9,12,14,18,23,26, be all positioned at the body region of proteins encoded, and the type of sudden change is relevant to the seriousness that courage becomes silted up.Nearly 1/3 case sudden change causes truncated protein and generates, and Liver immunity staining examine does not go out MDR3 glycoprotein.Another 2/3 case is missense mutation, mostly occurs at WalkerA and the B motif relating to ATP combination of high conservative.The change of these amino acid does not affect atpase activity and transport process, but causes MDR3 glycoprotein assembly defect in cell, functional defect.
PFIC1 type and PFIC2 type clinical phenotypes similar, by the detection to ATP8B1 and ABCB11 gene, contribute to making a definite diagnosis.PFIC is autosomal recessive hereditary diseases, infant siblings have the morbidity of 25%, 50% the possibility of carrying pathogenic mutation.Disease-causing gene detection is carried out to asymptomatic siblings and not only contributes to early discovery disease, early treatment.And, because various dcc gene has been studied clear all, so gene diagnosis is the most accurate.Sanger sequencing is generally acknowledged at present, and the gold standard of most of clinical molecular diagnosis project, specificity even can reach 100%, is the classical technology platform of molecular diagnosis aspect.Therefore adopt Sanger sequencing to carry out abrupt climatic change to the sequence of ATP8B1, ABCB11 and ABCB4, there is stability high, the advantage that specificity is good, can effectively avoid false-positive generation.
In order to provide reliable template to Sanger order-checking, first must for the feature of this project, set up a reliable and stable PCR system.This project PCR part two difficult points below sport technique segment exists: 1. this project needs to extract poba gene group DNA, owing to there is numerous uncontrollable factor in clinical samples logistics transportation, therefore often can not get superior in quality whole blood sample in Clinical Laboratory, also can there is difference between larger sample in the DNA quality therefore obtained.2. the exon number owing to detecting is more, and namely PCR object fragment is too much, causes PCR condition disunity.
Solvent temperature (Tm) is a very important parameter in PCR, primer and the temperature of complementary sequence when showing as double chain DNA molecule of 50%, necessary for setting PCR annealing temperature, suitable annealing temperature effectively can reduce non-specific binding, can also ensure that aim sequence is effectively annealed.So in the specific situation of guarantee, the Tm value of design primer is as far as possible close, the annealing temperature of PCR can be consistent, thus reach the unification of PCR condition.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of test kit and the detection method thereof that detect the transgenation of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease, this test kit can be used for the mutational site detecting ATP8B1 gene, ABCB4 gene, ABCB11 gene, specificity is good, highly sensitive.
A kind of test kit detecting the transgenation of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease of the present invention, described test kit comprises pcr amplification reaction reagent and the PCR primer for ATP8B1 gene, ABCB4 gene, ABCB11 gene amplification in sample DNA; 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。
Described pcr amplification reaction reagent comprises
archaeal dna polymerase, dNTPs, MgCl
2and reaction buffer.
A kind of detection method detecting the test kit of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease transgenation of the present invention, comprises the steps:
(1) gather blood sample to be measured, extract DNA;
(2) with this DNA for template, adopt be used for ATP8B1 gene in sample DNA, ABCB4 gene, ABCB11 gene amplification PCR primer carry out pcr amplification, obtain PCR reaction product, carry out sequencing analysis, determine whether there is base mutation.
PCR reaction conditions in described step (2) is: 95 DEG C of 5min; 95 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 40s, 15 circulations; 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 described step (2) is: pcr amplification reaction reagent 10 μ L, ddH
2o6 μ L, each 1 μ L of upstream and downstream primer, sample DNA 2 μ L.
beneficial effect
The present invention can be used for the mutational site detecting ATP8B1 gene, ABCB4 gene, ABCB11 gene, and specificity is good, highly sensitive; Can be used for clinical in Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease early discovery, the Subsidiary Index of clarifying a diagnosis in early days, reduce misdiagnosis rate and the treatment that avoids delay; Family member carries out examination can specify onset risk, and Prenatal Screening is also intervened, and can reduce the sickness rate of offspring's Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease, using method is simple, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is the order-checking peak figure of ATP8B1 (A), ABCB4 (B) and ABCB11 (C) genic mutation type.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
1. sample extracting
(1) extract the whole blood of 200 μ L, add 20 μ L Proteinase K Solution, mixing.
(2) add 200 μ L damping fluid GB, fully put upside down mixing, place 10 minutes for 56 DEG C, put upside down mixing therebetween for several times, solution strain is limpid, and brief centrifugation is to remove the globule of cap wall.
(3) add 200 μ L dehydrated alcohols, fully put upside down mixing, now may occur flocks, brief centrifugation is to remove the globule of cap wall.
(4) previous step gained solution and flocks are all added in an adsorption column CB3 (adsorption column puts into collection tube), centrifugal 30 seconds of 12000rpm (13400 × g), outwell the waste liquid in collection tube, adsorption column CB3 is put back in collection tube.
(5) in adsorption column CB3, add 500 μ L damping fluid GD (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12000rpm (13400 × g), outwells waste liquid, adsorption column CB3 is put into collection tube.
(6) in adsorption column CB3, add 600 μ L rinsing liquid PW (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12000rpm (13400 × g), outwells waste liquid, adsorption column CB3 is put into collection tube.
(7) in adsorption column CB3, add 600 μ L rinsing liquid PW, centrifugal 30 seconds of 12000rpm (13400 × g), outwells waste liquid.
(8) put back in collection tube by adsorption column CB3, centrifugal 2 minutes of 12000rpm (13400 × g), outwells waste liquid.Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
(9) adsorption column CB3 is proceeded in a clean 1.5mL centrifuge tube, the unsettled dropping 50 in middle part to adsorption film μ L elution buffer TE, room temperature places 2-5 minute, 12000rpm (13400 × g) centrifugal 2 minutes, by solution collection in centrifuge tube.
2. experimentation
(1) by sample number n (sample number=number of awaiting test sample+negative control 1+positive control 1) get pre-mixed PCR reaction system often pipe 20 μ L be sub-packed in reaction tubes.
(2) the above-mentioned sample to be tested handled well and feminine gender, positive control are respectively got 2 μ L and added respectively in reaction tubes, mixing, the low-speed centrifugal several seconds, carry out pcr amplification, concrete reaction system is as follows:
PCR reaction conditions, sees the following form:
3. sequencing analysis
ATP8B1 sequencing primer sequence
ABCB4 sequencing primer sequence
ABCB11 sequencing primer sequence
The concrete steps of 4.Sanger order-checking are as follows:
4.1 order-checking PCR:
4.1.1 preparation of reagents: according to the form below carries out, and operates on ice, is kept at-20 DEG C of refrigerators 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 | - |
4.2PCR product purification
4.2.1 take out point purification reaction liquid installed, well number accordingly at tube wall subscript.
4.2.2 getting 9ulPCR product adds in the pipe of corresponding numbering, mixing (noting producing bubble).
4.2.3 will mix sample according to PCR instrument on following reaction conditions, carry out enzyme purification amplification.
37℃→95℃→4℃
50′5′+∞
4.2.4 the sample that purifying is good is used for next step sequencing reaction, as the same day need not put freezen protective.
4.3 sequencing reaction
4.3.1 each sample does positive and negative two reactions, and mark finished writing by corresponding thin-walled tube.
4.3.2 every reaction system 5ul, BigDye mixed solution and 5P primer fixedly add 1ul, and template generally adds 2ul, and maximum is 3ul (according to the adjustment of PCR primer electrophoresis result), supplies 5ul with water.
4.3.3 first add the water of respective amount, then add corresponding 1ul primer and be added in water, then add the BigDye mixed solution of 1ul in the difference place of tube wall, finally add template.
4.3.4 lid upper tube cap low speed is of short duration centrifugal, and vortice mixes, and low speed is of short duration centrifugal again.
4.3.5 according to PCR instrument on following reaction conditions, 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 primer increased is carried out of short duration centrifugal.At the every adherent 125mMEDTA adding 2ul in pipe side, then add 15ul dehydrated alcohol the opposite side of pipe is adherent, vortex mixes.
4.4.2 use board-like whizzer, 4000rpm, centrifugal 30min, then 300rpm is inverted brief centrifugation, removes supernatant.
4.4.3 add 20 DEG C of precooling 75% ethanol of 50ul, vortex mixes, 4000rpm, 4 DEG C of centrifugal 15min.
4.4.4300rpm be inverted brief centrifugation, remove supernatant, be then positioned over room temperature, 15min, be evaporated completely ethanol.
4.4.5, after ethanol volatilizees completely, often pipe adds water and Hi-Di
tMeach 5ul, abundant vortex oscillation 1min., of short duration centrifugal rear standing 15min. makes order-checking product dissolve completely.
4.4.6 by Hi-Di
tMlysate is transferred in the corresponding position of 96 orifice plates.Put in PCR amplification instrument, 95 DEG C of denaturation 5min., be transferred to rapidly 4 DEG C of cooling 5min on ice chest.To 3500 sequenators, machine checks order and analyzes.
Data analysis: application sequencinganalysis software carries out sequence alignment analysis.The standard sequence retrieved in the sequence and NCBI that obtain checking order is compared, the gene order of confirmatory sample.
5. clinical samples comparison: preclinical Samples detection 30 example, test kit detected result coincidence rate 90%.
Therefore, use test kit of the present invention, adopt sequencing technologies, ATP8B1 gene, ABCB4 gene, ABCB11 transgenation are detected.By Auele Specific Primer and the fluorescently-labeled ddNTP of band, the gene mutation site that disease of depositing to Progressive symmetric erythrokeratodermia familial hepatic bile is relevant can be detected.The early discovery that may be used for clinical Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease, the Subsidiary Index of clarifying a diagnosis in early days, and the early screening method of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease patient.
Claims (5)
1. detect a test kit for Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease transgenation, it is characterized in that: described test kit comprises pcr amplification reaction reagent and the PCR primer for ATP8B1 gene, ABCB4 gene, ABCB11 gene amplification in sample DNA; 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 test kit detecting the transgenation of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease according to claim 1, is characterized in that: described pcr amplification reaction reagent comprises
archaeal dna polymerase, dNTPs, MgCl
2and reaction buffer.
3. the detection method detecting the test kit of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease transgenation as claimed in claim 1, comprises the steps:
(1) gather blood sample to be measured, extract DNA;
(2) with this DNA for template, adopt be used for ATP8B1 gene in sample DNA, ABCB4 gene, ABCB11 gene amplification PCR primer carry out pcr amplification, obtain PCR reaction product, carry out sequencing analysis, determine whether there is base mutation.
4. a kind of detection method detecting the test kit of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease transgenation according to claim 3, is characterized in that: the PCR reaction conditions in described step (2) is: 95 DEG C of 5min; 95 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 40s, 15 circulations; 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, 25 circulations; Last 72 DEG C of 10min.
5. a kind of detection method detecting the test kit of Progressive symmetric erythrokeratodermia familial hepatic bile alluvial disease transgenation according to claim 3, is characterized in that: the PCR reaction system in described step (2) is: pcr amplification reaction reagent 10 μ L, ddH
2o6 μ L, each 1 μ L of upstream and downstream primer, sample DNA 2 μ L.
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