CN113832148A - Rapid typing detection method for human SERPINB7 gene mutation - Google Patents
Rapid typing detection method for human SERPINB7 gene mutation Download PDFInfo
- Publication number
- CN113832148A CN113832148A CN202111091748.3A CN202111091748A CN113832148A CN 113832148 A CN113832148 A CN 113832148A CN 202111091748 A CN202111091748 A CN 202111091748A CN 113832148 A CN113832148 A CN 113832148A
- Authority
- CN
- China
- Prior art keywords
- seq
- probe
- sequence
- detection
- sites
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 72
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 16
- 101000836084 Homo sapiens Serpin B7 Proteins 0.000 title claims abstract description 10
- 102000050781 human SERPINB7 Human genes 0.000 title claims abstract description 6
- 239000000523 sample Substances 0.000 claims abstract description 104
- 239000000203 mixture Substances 0.000 claims abstract description 28
- 230000035772 mutation Effects 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims description 29
- 101150094135 SERPINB7 gene Proteins 0.000 claims description 13
- 238000012408 PCR amplification Methods 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 230000000171 quenching effect Effects 0.000 claims description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 229960003237 betaine Drugs 0.000 claims description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- -1 BHQ-3 Chemical compound 0.000 claims 1
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims 1
- 238000003556 assay Methods 0.000 claims 1
- 230000000295 complement effect Effects 0.000 claims 1
- 206010020649 Hyperkeratosis Diseases 0.000 abstract description 4
- 208000001126 Keratosis Diseases 0.000 abstract description 4
- 230000007918 pathogenicity Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 29
- 238000006243 chemical reaction Methods 0.000 description 16
- 239000012295 chemical reaction liquid Substances 0.000 description 13
- 150000007523 nucleic acids Chemical class 0.000 description 13
- 239000000243 solution Substances 0.000 description 11
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 239000000047 product Substances 0.000 description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 206010033554 Palmoplantar keratoderma Diseases 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 229960001484 edetic acid Drugs 0.000 description 5
- 201000008743 palmoplantar keratosis Diseases 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 125000006853 reporter group Chemical group 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000000630 rising effect Effects 0.000 description 3
- 102220034715 rs142859678 Human genes 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 206010015150 Erythema Diseases 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 101150033839 4 gene Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 206010053177 Epidermolysis Diseases 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical class NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 208000008454 Hyperhidrosis Diseases 0.000 description 1
- 208000005775 Parakeratosis Diseases 0.000 description 1
- 241000411545 Punargentus Species 0.000 description 1
- 229940122055 Serine protease inhibitor Drugs 0.000 description 1
- 101710102218 Serine protease inhibitor Proteins 0.000 description 1
- 102100025521 Serpin B7 Human genes 0.000 description 1
- 206010040844 Skin exfoliation Diseases 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- OGQICQVSFDPSEI-UHFFFAOYSA-N Zorac Chemical compound N1=CC(C(=O)OCC)=CC=C1C#CC1=CC=C(SCCC2(C)C)C2=C1 OGQICQVSFDPSEI-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 210000001361 achilles tendon Anatomy 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 210000003423 ankle Anatomy 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960004703 clobetasol propionate Drugs 0.000 description 1
- CBGUOGMQLZIXBE-XGQKBEPLSA-N clobetasol propionate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CCl)(OC(=O)CC)[C@@]1(C)C[C@@H]2O CBGUOGMQLZIXBE-XGQKBEPLSA-N 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000035618 desquamation Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000002683 foot Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 210000004247 hand Anatomy 0.000 description 1
- 206010072537 hereditary palmoplantar keratoderma Diseases 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000037315 hyperhidrosis Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 230000037378 palmoplantar hyperhidrosis Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 150000004492 retinoid derivatives Chemical class 0.000 description 1
- 102220047872 rs672601344 Human genes 0.000 description 1
- 102220034716 rs797044479 Human genes 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229960000565 tazarotene Drugs 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a rapid typing detection method for human SERPINB7 gene mutation, which comprises a series of primer probe compositions of different sites and a corresponding rapid typing detection method. The primer probe composition designed by the invention can detect four mutation sites of SERPINB7 genes, namely c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT, and has excellent specificity. By using a TaqMan detection method, the kit can specifically, accurately and quickly detect the pathogenicity gene of the long-island palm and toe keratosis, and is suitable for large-scale popularization and application.
Description
Technical Field
The invention belongs to the technical field of in-vitro nucleic acid detection, and relates to a rapid typing detection method for human SERPINB7 gene mutation, in particular to a primer probe composition for detecting SERPINB7 gene mutation and a rapid typing detection method.
Background
Long island type palmoplantar keratosis (NPPK, OMIM 615598) belongs to diffuse non-destructive palmoplantar keratosis, is an autosomal recessive hereditary palmoplantar keratosis (PPK), belongs to diffuse non-epidermolysis palmoplantar keratosis, and is the most common type of palmoplantar keratosis of Chinese Han population. The disease is reported in 1977 for the first time in Changdai, the incidence rate is relatively high, and the incidence rate is about 3.1/10000 in Chinese Han people. The causative gene of NPPK has been identified to date as serine protease inhibitor B7(serpinfamilyB, SERPINB 7).
The SERPINB7 gene is located at 18q21.3, and has 8 exons, the initiation codon is located at exon 2, and the termination codon is located at exon 8, and encodes 380 amino acids. The NPPK pathogenic mutations on SERPI NB7 genes are reported to be c.796C > T, c.218_219del AGinsTAAACTTT ACCT, c.455-1G > A, c.455G > T, c.650-653del CTGT, c.522-523insT, c.336+2T > G, c.122_127del TGGTCC, c.830C > T, c.382C > T, c.635delG, c.271delC and c.1136G > A, wherein the c.796C > T mutation frequency is the highest, and the carrying frequency of c.796C > T in the normal population in China is 6/197.
NPPK is clinically characterized in that mild keratosis is caused on the basis of skin erythema, and the clinical characteristics are that erythema, pimples, plaques, keratosis and desquamation appear on parts such as palms, wrists, soles, instep, ankles, achilles tendons and the like, the hyperhidrosis of hands and feet is accompanied easily with skin superficial fungal infection, and most patients are accompanied with palmoplantar hyperhidrosis and/or palmoplantar peculiar smell[2-3]. At present, no treatment method for radically curing NPPK (NPPK) is found, wherein the treatment methods can relieve the condition of a patient by using a retinoid medicament such as tretinoin ointment and tazarotene or a hormone ointment such as clobetasol propionate and combining a fungal infection with an antifungal medicament, but the disease is easy to relapse after the medicament is stopped[6]。
Currently, detection of mutations in SERPINB7 gene relies mainly on sanger sequencing. The basic principle of the method is that after PCR amplification, the obtained target gene fragment is subjected to sanger sequencing, and whether the pathogenic SERPINB7 gene mutation exists or not is analyzed according to the sequencing result. The method has the disadvantages of complex experimental operation, long time, corresponding professional knowledge required for result analysis and relatively high detection cost. Therefore, the real-time fluorescence quantitative PCR with high efficiency, rapidness and simple operation becomes the main method in the field of molecular diagnosis.
According to the data of NCBI dbSNP database, the kit detects 4 sites: the mutant allele frequencies of c.796C > T (rs142859678), c.522_523insT (rs672601344), c.650_653delCTGT (rs534014297), c.806_818delinsT (rs1157759655) in the normal east Asian population are respectively: 0.0119, 0.0032, 0.003, 0.0006, from which the rates of carriage of the four sites in the population were calculated to be about 3.7/100.
[1]Nagashima M.Handbook of Human Genetics[M].Tokyo:Igaku Shoin,1977:23-27.
[2]Kabashima K,Sakabe J,Yamada Y,et al.“Nagashima-type”keratosis as a novel entity in the palmoplantar keratoderma category[J].A rch Dermatol,2008,144(3):375-379.
[3]Kubo A,Shiohama A,Sasaki T,et al.Mutations in SERPINB7,encoding a member of the serine protease inhibitor superfamily,cause Nagas hima-type palmoplantar keratosis[J].Am J Hum Genet,2013,93(5):945-956.
[4]Zhang J,Zhang G,Ni C,et al.Nagashima-type palmoplantar ker atosis in a Chinese Han population[J].Mol Med Rep,2016,14(5):4049-4054.
[5] Daishan, Nannan vivid, Zhao hongshan, et al, Long island type palmoplantar keratosis SERPINB7 Gene mutation site research [ J ] Chinese and Western medicine integration dermatosis journal, 2017,16(2): 108-.
[6] Mutation analysis of SERPINB7 Gene in an example of Long island Palmar keratosis [ J ] Youjiang medicine, 2018,46(1):23-25.
Disclosure of Invention
The invention mainly aims to provide a primer probe composition of SERPINB7 gene mutation and a rapid typing detection method aiming at the defect that no product capable of being used for NPPK gene diagnosis exists at home and abroad at present, and a corresponding detection kit can be further developed on the basis of the primer probe composition and the method. In some embodiments, the detection kit developed according to the primer probe composition and the TaqMan probe method can specifically, accurately and quickly detect the pathogenicity gene of the long-island palm and toe keratosis, and is suitable for large-scale popularization and application.
In order to achieve the aim, the invention provides a primer probe composition for detecting the human SERPINB7 gene, which comprises a primer pair of at least one gene locus and a corresponding probe,
c.796C > T sites are: primers with sequences shown as SEQ ID No.1 and SEQ ID No.2, and probes with sequences shown as SEQ ID No.3 and/or SEQ ID No. 4;
c.522_523insT site: primers with sequences shown as SEQ ID No.5 and SEQ ID No.6, and probes with sequences shown as SEQ ID No.7 and/or SEQ ID No. 8;
c.650-653 delCTGT site was chosen as: primers with sequences shown as SEQ ID No.9 and SEQ ID No.10, and a probe with sequence shown as SEQ ID No. 12; or, primers with sequences shown in SEQ ID No.9 and SEQ ID No.11, and probe with sequence shown in SEQ ID No. 13;
c.806_818delinsT sites: the primers with the sequences shown in SEQ ID No.1 and SEQ ID No.14, and the probes with the sequences shown in SEQ ID No.15 and/or SEQ ID No. 16.
Preferably, the 5 'end of any one of the probes is connected with a fluorescent group, and the 3' end of any one of the probes is connected with a fluorescence quenching group.
Preferably, the fluorescent group attached to any one of the probes is independently selected from 1 of FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red or CY 3.
Preferably, the fluorescent group connected with the probe with the sequence shown as SEQ ID No.3 is FAM,
the sequence is shown as SEQ ID No.4, the fluorescent group connected with the probe is ROX,
the sequence is shown as SEQ ID No.7, the fluorescent group connected with the probe is CY5,
the sequence is shown as SEQ ID No.8, the fluorescent group connected with the probe is ROX,
the sequence is shown as SEQ ID No.12, the fluorescent group connected with the probe is VIC,
the sequence is shown as SEQ ID No.13, the fluorescent group connected with the probe is FAM,
the sequence is shown as SEQ ID No.15, the fluorescent group connected with the probe is CY5,
the sequence is shown as SEQ ID No.16, and the fluorescent group connected with the probe is VIC.
Preferably, the quenching group attached to any one of the probes is independently selected from 1 of MGB, BHQ-1, BHQ-2, BHQ-3 or TAMRA.
In another aspect, the invention provides a rapid typing detection method using the primer probe composition. Wherein the detected gene mutation sites are c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT sites of the SERPINB7 gene, and the used method is a TaqMan probe method.
The TaqMan probe method specifically comprises the following steps:
step 1: processing a blood sample, and processing an EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample by using a sample releasing agent to obtain a mixed solution containing nucleic acid to be detected;
step 2: selectively and specifically amplifying a target nucleic acid sequence by using the nucleic acid solution obtained in the step 1 as a template and a PCR amplification system containing the primer probe composition; wherein the target sequence is a nucleic acid sequence corresponding to each primer pair;
and step 3: and (3) measuring the fluorescence intensity amplified in the step (2) by using an instrument, carrying out quantitative analysis, and judging the genotypes of 4 pathogenic mutation sites on the SERPINB7 gene in the detection range of the sample to be detected.
The quantitative detection principle of the TaqMan probe method is as follows: when the probe is complete, no fluorescence can be detected, in the PCR extension process, the DNA polymerase hydrolyzes the probe and then emits a fluorescent signal, and the fluorescence increase value is in positive correlation with the amount of PCR products.
Preferably, the c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT sites need grouping detection, and the grouping detection is at least 2 groups. In practical operation, grouping detection can be carried out according to practical requirements, and wild type and mutant type of one or more sites can be detected in each grouping. It should be noted that the actual grouping detection needs to be adjusted according to the number of channels of the fluorescence detection instrument. When the number of fluorescence detection channels is 8, the wild type and the mutant type of each locus can be detected simultaneously; when the number of fluorescence channels is 4, detection should be performed in two groups.
Further preferably, the grouping detection is 2, 3, 4, 5, 6, 7, 8 groups.
Preferably, when the packet detection is 2 groups:
the first group of detection sites comprises a mutant type of a c.796C > T site, a wild type and a mutant type of a c.522_523insT site and a wild type of a c.650_653delCTGT site;
the second set of detection sites includes the wild type of the c.796C > T site, the mutant of the c.650-653 delCTGT site, the wild type of the c.806-818 delinsT site and the mutant.
When the grouped detection method is adopted, the detection times can be reduced, and the mutual interference between a plurality of primer pairs and corresponding detection probes can be avoided; and proved by verification, the primer probe composition is used for detecting the gene mutation of SERPINB7, and has good specificity and high sensitivity.
Preferably, the PCR amplification system in the TaqMan probe method comprises DNA polymerase, dNTP and Mg besides the primer probe composition2+、ROX Dye。
Preferably, the PCR amplification system further comprises Tris, 10 mM-500 mM KCl, 10 mM-500 mM (NH)4)2SO41 to 50 percent of glycerol, 0.001 to 1mg/mL of BSA, 0.1 to 10 percent of Tween 20, 1 to 100mM/mL of dithiothreitol, 0.1 to 3M of betaine and 0.1 to 10 percent of DMSO.
In the specific implementation process, the actual PCR amplification system comprises DNA polymerase, dNTP and Mg2+At least one of PCR buffer system, PCR intensifier and PCR stabilizer. The pH value of the PCR buffer system is 7.5-9.5 (at 25 ℃).
In certain embodiments, the above-described rapid sample release agent for EDTA anticoagulated whole blood sample includes, but is not limited to, erythrocyte lysate, DNA release promoter, detergent, proteinase K, guanidinium salt, and the like. The rapid sample release agent can be used for extraction-free rapid nucleic acid release.
In certain embodiments, the present invention can also be used for control analysis using the four-site mutation control and the normal control as described below. Wherein the mutation control is a plasmid containing the 4-locus mutant gene sequence, and the normal control is normal human genome DNA.
The invention has the following beneficial effects:
the primer probe composition for detecting four mutation sites of the SERPINB7 genes, namely c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT, is a TaqMan MGB probe specially used for optimizing the binding property of a mismatch primer design, and has excellent specificity. By using a TaqMan detection method, four pathogenic mutation sites can be rapidly typed and detected, and at least two groups of the four pathogenic mutation sites can be completely detected. The method is based on a general fluorescent quantitative PCR platform, has low equipment cost, high popularity, simple operation, easy interpretation of results and no pollution when the tube is closed in the whole process. In addition, the method does not need nucleic acid extraction, and is completed within 1.5h from sample treatment to detection result.
Detailed Description
Embodiments of the present application will be described in detail by examples, so that how to apply technical means to solve technical problems and achieve technical effects of the present application can be fully understood and implemented.
The raw materials and equipment used in the present application are all common raw materials and equipment in the field, and are all from commercially available products, unless otherwise specified. The methods used in this application are conventional in the art unless otherwise indicated.
There are many other possible embodiments of the present invention, which are not listed here, and the embodiments claimed in the claims of the present invention can be implemented.
"comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not excluding others. When used in defining compositions and methods, "consisting essentially of … …" is meant to exclude other elements having any significance to the combination of the stated objects. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed application. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transition terms are within the scope of the present application.
Example 1
The invention aims to provide a low-cost and high-efficiency composite PCR amplification system capable of genotyping 4 SNP sites on SERPINB7 gene causing parakeratosis palmaris et plantaris at the same time. The 4 common mutation sites on the SERPINB7 gene are c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT respectively. Wherein, the wild type gene sequences of the four sites of c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT are respectively shown as SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No. 20.
The gene sequences of the primer probe composition for detecting the SERPINB7 gene mutation are shown in Table 1. The primer pair probe composition of the multiplex PCR amplification system for 4 gene loci, as well as the fluorescein modification characteristics are also labeled in table 1. Wherein, the probe corresponding to each site is obtained by connecting a fluorescent group to the 5' end.
For the c.650_653delCTGT site, only one primer is shared by the wild type and the mutant, namely SEQ ID No. 9. Specifically, the primer pair corresponding to the wild type of the c.650_653delCTGT site is as follows: the sequences are shown as SEQ ID No.9 and SEQ ID No. 10. c.650-653 delCTGT site mutant corresponds to the primer pair: the sequences are shown as SEQ ID No.9 and SEQ ID No. 11.
Table 1.4 sites of 16 PCR amplification primers and fluorescein labeling characteristics
Table 2 shows the specific genotypes of the corresponding sites to be detected by the above probes.
TABLE 2
The primer probe composition shown above can be detected by a common TaqMan probe method. Each detection can be performed only aiming at one genotype of one site, and also can be performed on a plurality of sites at one time. It should be noted that, when a plurality of sites are simultaneously detected, the excitation wavelength of the fluorophore used for each probe in one detection should be different and have high resolution.
Example 2
For the convenience of measurement, this example designs a detection kit comprising the primer probe composition described above. It is to be understood that, although the detection method in example 2 is carried out in the form of a kit, in practice, it is only necessary to ensure that the same reaction system is used. Consistent results were also obtained using the same reagents outside the kit.
In this example, the reagents required to be contained in the typing test kit for palmar-shikayasis are shown in table 3.
TABLE 3
In a specific embodiment, in the detection reaction solution A and the detection reaction solution B, the final concentration of each primer can be selected within a range of 0.2 to 1.2. mu.M, and the final concentration of each probe can be selected within a range of 0.1 to 0.8. mu.M.
In this example, the final concentration of each primer was 0.6. mu.M and the final concentration of each probe was 0.35. mu.M in the detection reaction solution A and the detection reaction solution B.
Wherein the PCR reaction buffer solution also comprises the following components: tris, KCl 10-500 mM, NH 10-500 mM4)2SO41 to 50 percent of glycerol, 0.001 to 1mg/mL of BSA, 0.1 to 10 percent of Tween 20, 1 to 100mM/mL of dithiothreitol, 0.1 to 3M of betaine and 0.1 to 10 percent of DMSO.
Specific sequences corresponding to the primer probe compositions contained in the detection reaction solutions a and B are shown in table 4.
TABLE 4
The reagent is used for detecting a sample to be detected through a fluorescent quantitative PCR instrument, and the method comprises the following specific steps:
1. sample pretreatment
The kit does not need to extract and purify sample DNA before detection, and can be used for detection only after simple treatment.
The processing steps are as follows:
a) sucking 3 times of volume of precipitation treatment buffer solution of the pretreated human anticoagulated whole blood into a 0.2mL eight-connected tube, wherein the volume of the human anticoagulated whole blood is recommended to be 25 mu L, and the minimum volume of the human anticoagulated whole blood is not less than 10 mu L;
b) adding anticoagulated whole blood to be treated into a precipitation treatment buffer solution, blowing and sucking back and forth for several times by using a pipettor to completely dissolve the blood remained on the suction head, and violently shaking for 1min (fully shaking and uniformly mixing are very important); after 5 minutes at room temperature, the mixture was inverted several times, centrifuged at 7000rpm for 5 minutes, and the supernatant was aspirated off, leaving the precipitate for further processing.
c) Adding 80 μ L of nucleic acid release buffer solution into the precipitate, shaking for 1min, mixing to obtain nucleic acid solution for detection, and storing at-20 + -5 deg.C if not detected immediately;
the volume of an EDTA (ethylene diamine tetraacetic acid) anticoagulation whole blood sample required by the kit is 25 mu L, and the treated mixed solution can be directly used for the detection of the kit according to the sample volume required by the detection.
2. Sample detection
1) Taking out the kit which is stored at minus 15 to minus 25 ℃ in a dark place, and balancing the components of the kit to room temperature;
2) taking out a corresponding number of 96-well plates or PCR reaction tubes;
3) calculating and transferring a corresponding amount of reagent according to the reaction number in the current experiment, wherein the reaction number refers to the sample inspection amount plus 1 blank control (DNA is replaced by purified water), 1 mutation control and 1 normal control, and the rest of the reagent is stored under the condition of keeping out of the sun at the temperature of between 15 ℃ below zero and 25 ℃ below zero;
4) preparing a reaction system A and a reaction system B according to the scheme in the table 5;
table 5: preparation of amplification reaction tube A/B
5) The template solution refers to the sample mixed solution, the blank reference substance, the mutation reference substance and the normal reference substance which are pretreated in the step 1;
6) sealing a 96-well plate with a film or covering a PCR reaction tube cover, oscillating uniformly, centrifuging at 2000rpm for 10 seconds, and putting the product into a fluorescent quantitative PCR instrument;
7) PCR amplification was performed according to the conditions of table 6:
table 6: amplification procedure
And after the setting is finished, saving the file and operating the reaction program.
3. Results analysis (please refer to the instruction of each apparatus, and the ABI series apparatus is used as an example in the following analysis)
After the reaction is finished, the instrument automatically stores the result, and after the image is analyzed, the threshold value of the fluorescence signal is adjusted until all fluorescence channels corresponding to the wild type in the detection result of the mutation comparison product have no signal, and all fluorescence channels corresponding to the mutant type in the detection result of the wild type comparison product have no detection signal. Taking the ABI 7500 type fluorescence quantitative PCR instrument as an example, an ideal detection result can be obtained when the fluorescence signal threshold is about 25000.
4. Quality control
The results of the mutation control, normal control and blank control tests should meet the following requirements, otherwise the test is regarded as invalid.
1) The detection results of the reaction liquid A and the reaction liquid B in the kit are shown in the following table 7:
table 7: comparison table for detecting fluorescence channel and detected gene in reaction liquid A/B
2) Blank control:
no signal exists before the Ct value of any detection channel in the detection results of the reaction liquid A and the reaction liquid B is 38. If the signal rises and the Ct value is less than or equal to 38, the experimental result is invalid, and re-experiment is recommended.
3) The mutant controls need to meet both:
a) the Ct value of a channel corresponding to any wild type in the detection results of the reaction liquid A and the reaction liquid B has no signal before 38;
b) obvious signal rising is generated in the channel corresponding to any mutant in the detection results of the reaction liquid A and the reaction liquid B, and the Ct value is less than or equal to 38.
If the signal of any channel in a) rises and the Ct value is less than or equal to 38, or no signal rises or a signal rises but the Ct value is more than 38 in any channel in b), the experimental result is invalid, and the re-experiment is recommended.
4) The normal reference substance needs to satisfy both:
c) the Ct value of a channel corresponding to any mutant in the detection results of the reaction liquid A and the reaction liquid B has no signal before 38;
d) obvious signal rising is generated in the channel corresponding to any wild type in the detection results of the reaction liquid A and the reaction liquid B, and the Ct value is less than or equal to 38.
If any channel signal in c) rises and Ct value is less than or equal to 38, or no signal rises in any channel in d), or signal rises but Ct value is more than 38, the experimental result is invalid, and re-experiment is recommended.
5. Interpretation of results
Under the condition that the quality control product is normal, the result is interpreted as follows:
if any detection channel in the sample to be detected has obvious signal rising and Ct value is less than or equal to 38, the detection result of the corresponding target gene of the detection channel is positive.
The detection principle is as follows: the Taqman probe method works on the principle that the 5 '→ 3' exonuclease activity of Taq enzyme is utilized. As the 5 'end of the probe is labeled with a fluorescence reporter group (such as FAM) and the fluorescence quenching group MGB is labeled close to the 3' end, an energy transfer structure is formed between the two. Therefore, when the probe is kept intact, the fluorescence signal excited by the 5 'end fluorescence reporter group is absorbed or inhibited by the 3' end quenching group, and no change of the fluorescence signal occurs. When the target gene exists in the PCR reaction system, a specific nucleic acid fragment is amplified, the fluorescent probe hybridizes with the target gene according to the base pairing principle, when the PCR enters an extension (replication) stage, Taq enzyme extends from the 3 ' end of the primer along a DNA template along with a new strand, and when the Taq enzyme extends to a position where the probe is combined, the probe is cut off under the action of exonuclease activity at the 5 ' → 3 ' end of the Taq enzyme. At this time, the energy transfer structure between the fluorescent reporter group and the quencher group is broken, the quenching effect of the quencher group is released, and the fluorescent signal of the fluorescent reporter group is released. For each replication of a specific nucleic acid fragment in the PCR reaction, one probe is cleaved with the release of a fluorescent signal. Along with the accumulation of the fluorescence signal, the fluorescence signal can be detected by a fluorescence PCR instrument. In contrast, when the probe sequence is not matched with the target gene sequence, the probe cannot be combined with the target sequence, the specific probe cannot be cut off in the PCR extension process, and a corresponding fluorescent signal cannot occur naturally.
After two SERPINB7 gene mutation samples (the specific genotypes are shown in Table 8) are treated, the nucleic acid solution is repeatedly detected for 10 times by using the kit, the detection result is positive and accords with the corresponding genotype, and the precision of the Ct value is less than or equal to 5.0%, which indicates that the kit has good repeatability.
Table 8: 2 examples of Gene mutation samples corresponding to genotypes
The nucleic acid solutions of 6 clinically diagnosed SERPINB7 gene mutation samples (the specific genotypes are shown in Table 9) are respectively detected by the kit, and the detection results are consistent with the clinical results, which shows that the kit has better accuracy and specificity.
Table 9: 6 examples of Gene mutation samples corresponding to genotypes
Sample numbering | Gene locus | Genotype(s) |
SER-01 | c.796C>T | Homozygous mutations |
SER-02 | c.522_523insT | Homozygous mutations |
SER-03 | c.796C>T | Heterozygous mutations |
SER-04 | c.522_523insT | Heterozygous mutations |
SER-05 | c.806_818delinsT | Heterozygous mutations |
SER-06 | c.650_653delCTGT | Heterozygous mutations |
The details not described in the specification of the present application belong to the common general knowledge of those skilled in the art.
In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect.
It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.
The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.
Sequence listing
<120> rapid typing detection method for human SERPINB7 gene mutation
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgtacctttc agaatctaat ggaat 25
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctcaacatac ttagaggtca tgca 24
<210> 3
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
accaatccaa ggtga 15
<210> 4
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cctatccaag gcga 14
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtgaaggtgg cataagctca tct 23
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
caatttatgg tttcgcttct g 21
<210> 7
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acacagcatt cacc 14
<210> 8
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cacaagcatt cacc 14
<210> 9
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cagtcgccat gatgcatca 19
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
agcagaacgt acatgtttat gcc 23
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggacaggaag ttcaatttgt ta 22
<210> 12
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ttgtctgtta ttgagg 16
<210> 13
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ttgttattga ggaccc 16
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
attcttctct atgaagaact gagga 25
<210> 15
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cctctaagta tgttgagg 18
<210> 16
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cgaatgacct tggtatt 17
<210> 17
<211> 146
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ttacagattg aaaacaaacg tacctttcag aatctaatgg aatggaccta tccaaggcga 60
atgacctcta agtatgttga ggtatttttt cctcagttct tcatagagaa gaattatgaa 120
atgaaacaat atttgagagc cctagg 146
<210> 18
<211> 125
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atttgaaatg gcaatttatg gtttcgcttc tggtgaaggc tgattgccac ttgcctttga 60
agtacacagc attcaccagc accattacag cagatgagct tatgccacct tcaccaatca 120
cgttc 125
<210> 19
<211> 130
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gggaaggcag tcgccatgat gcatcaggac aggaagttca atttgtctgt tattgaggac 60
ccatcaatga agattcttga gctcagatac aatggtggca taaacatgta cgttctgctg 120
cctgagaatg 130
<210> 20
<211> 146
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ttacagattg aaaacaaacg tacctttcag aatctaatgg aatggaccta tccaaggcga 60
atgacctcta agtatgttga ggtatttttt cctcagttct tcatagagaa gaattatgaa 120
atgaaacaat atttgagagc cctagg 146
<210> 21
<211> 146
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ttacagattg aaaacaaact gacctttcag aatctaatgg aatggaccaa tccaaggtga 60
atgacctcta agtatgttga ggtatttttt cctcagttca agatagagaa gaattatgaa 120
atgaaacaat atttgagagc cctagg 146
<210> 22
<211> 126
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atttgaaatg gcaatttatg gtttcgcttc tggtgaaggc tgattgccac ttgcctttga 60
agtacacaag cattcaccag caccattaca gcagatgagc ttatgccacc ttcaccaatc 120
acgttc 126
<210> 23
<211> 126
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gggaaggcag tcgccatgat gcatcaggac aggaagttca atttgttatt gaggacccat 60
caatgaagat tcttgagctc agatacaatg gtggcataaa catgtacgtt ctgctgcctg 120
agaatg 126
<210> 24
<211> 134
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttacagattg aaaacaaacg tacctttcag aatctaatgg aatggaccta tccaaggcga 60
atgaccttgg tattttttcc tcagttcttc atagagaaga attatgaaat gaaacaatat 120
ttgagagccc tagg 134
Claims (10)
1. A primer probe composition for detecting human SERPINB7 gene mutation is characterized by comprising a primer pair of at least one gene locus and a sequence of a corresponding probe, or a complementary sequence:
c.796C > T sites are: primers with sequences shown as SEQ ID No.1 and SEQ ID No.2, and probes with sequences shown as SEQ ID No.3 and/or SEQ ID No. 4;
c.522_523insT site: primers with sequences shown as SEQ ID No.5 and SEQ ID No.6, and probes with sequences shown as SEQ ID No.7 and/or SEQ ID No. 8;
c.650-653 delCTGT site was chosen as: primers with sequences shown as SEQ ID No.9 and SEQ ID No.10, and a probe with sequence shown as SEQ ID No. 12; or, primers with sequences shown in SEQ ID No.9 and SEQ ID No.11, and probe with sequence shown in SEQ ID No. 13;
c.806_818delinsT sites: the primers with the sequences shown in SEQ ID No.1 and SEQ ID No.14, and the probes with the sequences shown in SEQ ID No.15 and/or SEQ ID No. 16.
2. The primer probe composition of claim 1, wherein a fluorescent group is attached to the 5 'end of any of said probes and a fluorescence quenching group is attached to the 3' end of any of said probes.
3. The primer probe composition of claim 2, wherein the fluorescent group attached to any one of said probes is independently selected from 1 of FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red, or CY 3.
4. The primer probe composition of claim 3, wherein:
the sequence is shown as SEQ ID No.3, the fluorescent group connected with the probe is FAM,
the sequence is shown as SEQ ID No.4, the fluorescent group connected with the probe is ROX,
the sequence is shown as SEQ ID No.7, the fluorescent group connected with the probe is CY5,
the sequence is shown as SEQ ID No.8, the fluorescent group connected with the probe is ROX,
the sequence is shown as SEQ ID No.12, the fluorescent group connected with the probe is VIC,
the sequence is shown as SEQ ID No.13, the fluorescent group connected with the probe is FAM,
the sequence is shown as SEQ ID No.15, the fluorescent group connected with the probe is CY5,
the sequence is shown as SEQ ID No.16, and the fluorescent group connected with the probe is VIC.
5. The primer-probe composition of claim 2, wherein the quencher group attached to any one of the probes is independently selected from the group consisting of MGB, BHQ-1, BHQ-2, BHQ-3, and TAMRA.
6. A rapid typing detection method using the primer probe composition according to any one of claims 1 to 5, wherein the mutation sites of the genes to be detected are c.796C > T, c.522-523insT, c.650-653 delCTGT and c.806-818 delinsT sites of SERPINB7 gene, and the method is TaqMan probe method.
7. The rapid typing detection method according to claim 6 wherein the c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT sites are grouped for detection, and the group detection is at least 2 groups.
8. The rapid typing detection method according to claim 7, wherein when the packet detection is 2 groups:
the first group of detection sites comprises a mutant type of a c.796C > T site, a wild type and a mutant type of a c.522_523insT site and a wild type of a c.650_653delCTGT site;
the second set of detection sites includes the wild type of the c.796C > T site, the mutant of the c.650-653 delCTGT site, the wild type of the c.806-818 delinsT site and the mutant.
9. The rapid typing detection method as claimed in claim 8, wherein the PCR amplification system in the TaqMan probe method comprises DNA polymerase, dNTP, Mg in addition to the primer probe composition2+、ROX Dye。
10. The rapid typing assay method according to claim 9, wherein the PCR amplification system further comprises Tris, 10 mM-500 mM KCl, 10 mM-500 mM (NH)4)2SO41 to 50 percent of glycerol, 0.001 to 1mg/mL of BSA, 0.1 to 10 percent of Tween 20, 1 to 100mM/mL of dithiothreitol, 0.1 to 3M of betaine and 0.1 to 10 percent of DMSO.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111091748.3A CN113832148B (en) | 2021-09-17 | 2021-09-17 | Quick typing detection method for human SERPINB7 gene mutation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111091748.3A CN113832148B (en) | 2021-09-17 | 2021-09-17 | Quick typing detection method for human SERPINB7 gene mutation |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113832148A true CN113832148A (en) | 2021-12-24 |
CN113832148B CN113832148B (en) | 2024-03-29 |
Family
ID=78959726
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111091748.3A Active CN113832148B (en) | 2021-09-17 | 2021-09-17 | Quick typing detection method for human SERPINB7 gene mutation |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113832148B (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070072798A1 (en) * | 2005-07-12 | 2007-03-29 | Oy Jurilab Ltd | Method for treatment of cardiovascular and metabolic diseases and detecting the risk of the same |
CN110577993A (en) * | 2019-09-30 | 2019-12-17 | 苏州乾康基因有限公司 | Primer group, probe group, kit and application for detecting allergy-related gene mutation |
-
2021
- 2021-09-17 CN CN202111091748.3A patent/CN113832148B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070072798A1 (en) * | 2005-07-12 | 2007-03-29 | Oy Jurilab Ltd | Method for treatment of cardiovascular and metabolic diseases and detecting the risk of the same |
CN110577993A (en) * | 2019-09-30 | 2019-12-17 | 苏州乾康基因有限公司 | Primer group, probe group, kit and application for detecting allergy-related gene mutation |
Non-Patent Citations (3)
Title |
---|
J YIN 等: ""New and Recurrent SERPINB7 Mutations in Seven Chinese Patients with Nagashima-Type Palmoplantar Keratosis"", 《JOURNAL OF INVESTIGATIVE DERMATOLOGY》, vol. 134, pages 2269 - 2272 * |
刘成 等: ""长岛型掌跖角化病一家系丝氨酸蛋白酶 抑制剂B7 基因的突变分析"", 《皮肤性病诊疗学杂志》, vol. 25, no. 1, pages 8 - 11 * |
戴珊 等: ""长岛型掌跖角化病:SERPINB7基因突变位点研究"", 《中国中西医结合皮肤性病学杂志》, vol. 16, no. 2, pages 108 - 112 * |
Also Published As
Publication number | Publication date |
---|---|
CN113832148B (en) | 2024-03-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN114085903B (en) | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof | |
CN112063747B (en) | Primer probe group and kit for rapidly and efficiently detecting candida otophylla based on fluorescence PCR technology and application of primer probe group and kit | |
US20130078631A1 (en) | Probe, and polymorphism detection method using the same | |
EP2497835B1 (en) | Probe for detecting polymorphism in CYP3A gene, method of detecting polymorphism, method of evaluating drug efficacy, and reagent kit for detecting polymorphism | |
CN112941210A (en) | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid | |
CN111118138A (en) | Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR | |
CN108060263B (en) | Primer probe combination and fluorescent quantitative PCR kit for simultaneously detecting three cryptococci | |
WO2008077330A1 (en) | Taqman mgb probe for detecting maternal inherited mitochondrial genetic deafness c1494t mutation and its usage | |
CN106498028B (en) | Diagnostic method and kit for T790M mutation of EGFR | |
WO2008077329A1 (en) | Probe for detecting maternal inherited mitochondrial genetic deafness a1555g mutation and its usage | |
CN113502335A (en) | Molecular marker related to sheep growth traits and application thereof | |
CN113801933B (en) | Detection kit for rapid typing of human SERPINB7 gene mutation | |
CN113832148A (en) | Rapid typing detection method for human SERPINB7 gene mutation | |
US20190292586A1 (en) | Method of quantifying mutant allele burden of target gene | |
EP2653560B1 (en) | Probe, and polymorphism detection method using the same | |
CN113604589B (en) | Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor | |
EP2520665B1 (en) | Probe, polymorphism detection method, method of evaluating drug efficacy or tolerance, and reagent kit | |
CN112280852B (en) | SMN1 gene mutation detection kit and application thereof | |
CN110423807B (en) | Primer combination and kit for detecting deletion type alpha-thalassemia | |
CN110951858A (en) | Primer-probe combination for guiding detection of genes related to glibenclamide drug personalized administration, kit and application | |
CN110819709A (en) | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) | |
US20140087380A1 (en) | Mutation Detection Probe, Mutation Detection Method, Method of Evaluating Drug Efficacy, and Mutation Detection Kit | |
CN110923312A (en) | Real-time fluorescent PCR method for detecting rs762551 site of CYP1A2 gene and primer probe combination thereof | |
JP2004236655A (en) | Method for simply detecting genetic polymorph and reagent for detecting the same | |
RU2804110C1 (en) | Set of oligonucleotide primers and probes for determining alleles of the rs55986091 polymorphism and method for its use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |