CN104120167A - Method and kit for detecting mutation of PDS gene IVS7-2A>G by using fluorescence PCR technology - Google Patents
Method and kit for detecting mutation of PDS gene IVS7-2A>G by using fluorescence PCR technology Download PDFInfo
- Publication number
- CN104120167A CN104120167A CN201310142823.3A CN201310142823A CN104120167A CN 104120167 A CN104120167 A CN 104120167A CN 201310142823 A CN201310142823 A CN 201310142823A CN 104120167 A CN104120167 A CN 104120167A
- Authority
- CN
- China
- Prior art keywords
- ivs7
- pds gene
- probe
- wild
- type probe
- 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.)
- Pending
Links
- 101150108119 PDS gene Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 30
- 230000035772 mutation Effects 0.000 title claims abstract description 15
- 238000005516 engineering process Methods 0.000 title abstract description 5
- 239000000523 sample Substances 0.000 claims abstract description 92
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 230000003321 amplification Effects 0.000 claims abstract description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 15
- 239000003086 colorant Substances 0.000 claims abstract description 7
- 238000003908 quality control method Methods 0.000 claims description 35
- 238000012360 testing method Methods 0.000 claims description 24
- 230000008859 change Effects 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 238000013461 design Methods 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 239000007850 fluorescent dye Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 238000003556 assay Methods 0.000 claims description 6
- 239000013600 plasmid vector Substances 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 230000000869 mutational effect Effects 0.000 claims description 5
- 241000972773 Aulopiformes Species 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- 235000019515 salmon Nutrition 0.000 claims description 4
- -1 dNTP Substances 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 210000001776 amniocyte Anatomy 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 claims description 2
- 210000003296 saliva Anatomy 0.000 claims description 2
- 239000002585 base Substances 0.000 claims 2
- 239000000463 material Substances 0.000 claims 2
- 239000003513 alkali Substances 0.000 claims 1
- 206010011878 Deafness Diseases 0.000 abstract description 11
- 210000002905 vestibular aqueduct Anatomy 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 208000011580 syndromic disease Diseases 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000003745 diagnosis Methods 0.000 abstract 3
- 206010010356 Congenital anomaly Diseases 0.000 abstract 1
- 239000000969 carrier Substances 0.000 abstract 1
- 230000035935 pregnancy Effects 0.000 abstract 1
- 239000013062 quality control Sample Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 description 31
- 108020004414 DNA Proteins 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 238000007400 DNA extraction Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102100035278 Pendrin Human genes 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000035558 fertility Effects 0.000 description 3
- 229910001425 magnesium ion Inorganic materials 0.000 description 3
- 206010025482 malaise Diseases 0.000 description 3
- 230000007018 DNA scission Effects 0.000 description 2
- 241001643597 Evas Species 0.000 description 2
- 102000011384 Pendrin Human genes 0.000 description 2
- 108050001616 Pendrin Proteins 0.000 description 2
- 108091006507 SLC26A4 Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 231100000895 deafness Toxicity 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 208000016354 hearing loss disease Diseases 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000370738 Chlorion Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 206010011882 Deafness congenital Diseases 0.000 description 1
- 206010011891 Deafness neurosensory Diseases 0.000 description 1
- 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 1
- 206010014016 Ear malformation Diseases 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102100037156 Gap junction beta-2 protein Human genes 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 101000954092 Homo sapiens Gap junction beta-2 protein Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 208000009966 Sensorineural Hearing Loss Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000003027 ear inner Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 231100000879 sensorineural hearing loss Toxicity 0.000 description 1
- 208000023573 sensorineural hearing loss disease Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 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 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative 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
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method and a kit for detecting mutation of PDS gene IVS7-2A>G by using fluorescence PCR technology. The method comprises: aiming at a PDS IVS7-2A>G mutation site, designing a specific mutation type probe and a wild type probe and respectively marking with different colors, designing amplimers at two sides of the mutation site; and performing amplification on a to-be detected sample by using a PCR reaction system containing the wild type probe, the mutation type probe and the amplimers, and comparing the fluorescence curve mode with that of a quality-control sample to determine whether the sample has PDS gene IVS7-2A>G mutation. The method and the kit can provide clinical reference for disease cause diagnosis of deaf patient, especially deaf patients with enlarge vestibular aqueduct syndrome, also can be used to screen whether newborns and couples in pregnancy are carriers, and provides help for deaf antenatal diagnosis and newborn congenital deaf cause diagnosis. The kit has the characteristics of closed-tube high-flux automatic operation and analysis, and is especially usable in clinic laboratories.
Description
Technical field
The invention belongs to technique of gene detection field, particularly a kind of real time pcr of probe specificity detects method and the test kit thereof of deaf gene PDS IVS7-2A > G sudden change.
Background technology
Deafness is to cause the modal disease of communication obstacle, and the sickness rate of newborn infant's deafness is 0.1%～0.3%.China hearing and speech handicapped person is very many, and with annual newborn 30,000 deaf youngsters' speed increment, China's accumulative total approximately has 300,000 couples of at least one deaf youngsters' of fertility the couple at child-bearing age to face the selection of fertility again.The deaf reason occurring, whether there is heredity, whether safety is the problem that deaf family is very concerned about in fertility next time.Large vestibular aqueduct syndrome (Enlarge vestibular aqueduct syndrome.EVAS) is the recessive hereditary dysaudia disease that a kind of sickness rate is higher.Imaging examination data shows in children's patients with sensorineural hearing loss, and large vestibular aqueduct is modal a kind of inner ear malformations, and in hereditary deafness crowd, sickness rate is about 1%～1.3%, even can reach 7% in certain areas.The SLC26A4 assignment of genes gene mapping is in 7q31, and its mRNA total length 4930bp, has 21 exons. and open reading frame 2343bp, through exon 2 and exon 21,780 amino acid whose protein Pendrin encode.Pendrin is mainly made up of hydrophobic amino acid, belongs to ion transport body family, research show its function main with the feature of iodine chlorion transhipment about suddenling change.In EVAS patient, modal SLC26A4 transgenation is IVS7-2A > G.Chinese population is carried out to the general examination of IVS7-2A > G sudden change, can reduce the natality of large vestibular aqueduct infant.
The method of carrying out at present detection in Gene Mutation has tens of kinds.Gold standard method is PCR product direct sequencing.PCR product direct sequencing is current most widely used general, more a kind of method, but the method needs expensive plant and instrument and professional personnel to operate, and wastes time and energy, and is difficult to clinically promote.Gene chips has advantages of fast high-flux, but operation is upper complicated, and step is many, and cost height is also difficult for universal.With Restriction fragment length polymorphism (RFLP) detect the method for transgenation have advantages of with low cost, but process is loaded down with trivial details, the time is long to be also not suitable for popularizing.Disclosed patent (patent No. 200610058282.6) is according to PDS gene IVS7-2 mutational site A > G, design and can within the scope of upstream and downstream 1-13 base including this site, introduce the primer pair that any one Substitution suddenlys change, thereby can obtain the new restriction endonuclease sites that can differentiate A > G site in amplified production, the method needs follow-up enzyme to cut and the process such as electrophoresis, is not suitable for clinical expansion.Patent 201010599385.X, adopts Auele Specific Primer and probe, and by mononucleotide elongation technology, in conjunction with detections that suddenly change of micro-array chip technology, complexity is gone up in the method operation, and instrument costliness, is also difficult for penetration and promotion.
Present method adopts a kind of real-time fluorescence polymerase chain reaction technique (FQ-PCR) to detect the method for PDS gene IVS7-2A > G sudden change, for PDS gene IVS7-2A > G site design specificity wild-type probe and saltant type probe, wild-type and saltant type probe are labeled respectively the fluorescence of different colours, simultaneously at PDS gene IVS7-2 site two flank sequence area design specificity amplification primers, for detecting sample, the fluorescence color that produces in pcr amplification from saltant type probe according to wild-type different come judgement sample whether there is sudden change.It is simple that the method has short, high-throughput of time, stopped pipe automated operation, interpretation of result, detects the advantage that Single locus sudden change needs 1 PCR reaction tubes to realize.Being applicable to clinical labororatory uses.
Summary of the invention
The real-time fluorescence PCR detection method and the test kit that the object of the present invention is to provide a kind of PDS gene IVS7-2A > G sudden change, can be used for the detection of clinical sample.
Concrete technological line of the present invention is:
1) the 7th intron second-to-last base (the IVS7-2A > G) site of PDS gene extron 7-8 region (SEQ ID NO:5) design wild-type probe and saltant type probe, its length is the nucleotide sequence between 10-30bp.Two kinds of probes all cover PDS gene IVS7-2 site, and wherein wild-type probe (SEQ ID NO:3) is for PDS gene IVS7-2 wild-type site, and saltant type probe (SEQ ID NO:4) is for PDS gene IVS7-2 mutational site.
2) probe 5 ' is held the similar luminophores such as mark fluorescent generation group FAM or HEX, the similar quenching groups such as 3 ' end mark fluorescent quenching group TAMRA or BHQ1.Probe sequence is not limited to the concrete nucleotide sequence in this specification sheets, can be that any a section of covering PDS gene IVS7-2 site meets the nucleotide sequence that probe requires.The fluorescence generation group of probe 5 ' end mark and corresponding 3 ' end fluorescent quenching group thereof can be any in the art available fluorophors, and as FAM, HEX can be replaced by other fluorophors or dyestuff, as VIC, and JOE, CY5 etc.Can use conventional Taqman probe at present, can be also the LNA-Taqman probe through modifying on general T aqman probe basis, the relevant probe that improvement is modified on Taqman probe basis that MGB-Taqman probe or prior art can realize.
3) PDS gene IVS7-2 site upstream design forward amplimer, design reverse amplimer in downstream, its length is the nucleotide sequence between 10-30bp, the nucleotide fragments that can to amplify the length that contains PDS gene IVS7-2 site be 50-700bp, preferably, forward primer (SEQ ID NO:1) and reverse primer (SEQ ID NO:2) are for amplifying the specificity nucleotide sequence of the 60-150bp fragment that contains PDS gene IVS7-2 site.
4) preparation is suitable for the reaction system of pcr amplification.Nucleic acid amplification reaction system comprises: hot resistant DNA polymerase, dNTP, UNG enzyme, the damping fluid that contains Mg ion, forward primer (SEQ ID NO:1), reverse primer (SEQ ID NO:2), wild-type probe (SEQ ID NO:3), saltant type probe (SEQ ID NO:4).
5) from sample to be tested, extract DNA, add reaction system, carry out quantitative fluorescent PCR reaction.
6) show according to the different colours of wild-type, saltant type probe mark the genotype of determining sample.While only having the color demonstration of wild-type probe, sample is wild-type, otherwise is saltant type, if two kinds of colors all exist, sample is heterozygous.
PDS gene IVS7-2A > G mutation detection kit provided by the invention mainly comprises following component: PCR reaction solution, negative quality control product, positive quality control product, DNA cleavage liquid, erythrocyte cracked liquid and separation are also concentrated the packing box of packing these reagent bottles or pipe.
The present invention realizes according to following principle, in order to detect PDS (SEQ ID NO:5) gene IVS7-2A > G site, design covers saltant type probe and the wild-type probe in IVS7-2A > G site, wild-type probe can only be mated completely with wild-type sample template, saltant type probe can only mate completely with saltant type sample template, because saltant type probe is different from the fluorescence report group of wild-type probe 5 ' end mark, be the different fluorescence of the two emission wavelength, on fluorescent PCR instrument, recorded respectively and reflected with different colours or different marks in analysis software.
According to a preferred embodiment of the present invention, in test kit, PCR reaction solution contains 1XPCR Buffer, dNTPs, Mg2+, Taq enzyme, deionized water, forward primer (SEQ ID NO:1), reverse primer (SEQ ID NO:2), wild-type probe (SEQ ID NO:3), saltant type probe (SEQ ID NO:4).
Forward primer (SEQ ID NO:1): 5 '-GAATTATTAAAACCAATGGAGTTT-3 '
Reverse primer (SEQ ID NO:2): 5 '-GCATTCATTAAAAAAACCAGGTTGG-3 '
Wild-type fluorescent probe (SEQ ID NO:3): 5 ' FAM-TTGTTTTATTTCAGACGATAATT-BHQ13 '
Saltant type fluorescent probe (SEQ ID NO:4): 5 ' HEX-TTGTTTTATTTCGGACGATAATT-BHQ13 '
According to a preferred embodiment of the invention, in test kit, also can only add saltant type probe, in the time that template is saltant type, the fluorescence that saltant type probe is launched finally can be detected, thereby definite sample contains mutational site, in the time that template is wild-type, the fluorescence that saltant type probe is launched finally can not be detected, thereby definite sample does not contain mutational site.In theory, in an individual system, can only add saltant type probe, but in the time adding wild-type probe, can play the double insurance effect of reverse correction simultaneously, make result more accurately and reliably.Only add PCR reaction solution in the test kit of saltant type probe to contain 1XPCRBuffer, dNTPs, Mg2+, Taq enzyme, deionized water, forward primer (SEQ ID NO:1), reverse primer (SEQ ID NO:2), saltant type probe (SEQ ID NO:4).
Forward primer (SEQ ID NO:1): 5 '-GAATTATTAAAACCAATGGAGTTT-3 '
Reverse primer (SEQ ID NO:2): 5 '-GCATTCATTAAAAAAACCAGGTTGG-3 '
Saltant type fluorescent probe (SEQ ID NO:4): 5 ' HEX-TTGTTTTATTTCGGACGATAATT-BHQ13 '
According to a preferred embodiment of the invention, the positive quality control product in test kit is to be DNA or corresponding PCR product or the plasmid vector of tissue, blood or the clone extraction of PDS gene IVS7-2A > G homozygous mutation type through order-checking assay; Negative quality control product is distilled water, pure water, physiological saline or salmon sperm dna, can be also DNA or corresponding PCR product or the corresponding plasmid vector of tissue, blood or the clone extraction of PDS gene IVS7-2A > G wild-type for process order-checking assay;
According to a preferred embodiment of the invention, blood, saliva, amniocyte or other tissue of the detected person that in test kit, target nucleic acid sample to be detected obtains since various approach etc.
According to a preferred embodiment of the invention, the DNA extraction liquid in test kit mainly comprises Chelex-100.Also can use the DNA of other test kit or method extraction as the template of carrying out fluorescent PCR.
According to a preferred embodiment of the invention, in test kit, for the standard of judging detection method validity be: each detection is all used PCR reaction solution to detect respectively positive quality control product and negative quality control product, for positive quality control product, the color fluorescence curve producing according to saltant type probe is as positive quality control standard, and the Ct value of positive quality control product should be less than 30.For DNA or corresponding PCR product or the negative quality control product of corresponding plasmid vector to extract as the GJB2 235 wild homozygous tissues in site, blood or clone through order-checking assay, the color fluorescence curve producing according to wild-type probe is as negative quality control standard, and the Ct value of negative quality control product should be less than 30; For the negative quality control product with distilled water, pure water, physiological saline or salmon sperm dna, the Ct value of the color fluorescence curve that wild-type and saltant type probe produce all should be greater than 35.
According to a preferred embodiment of the invention, can be according to the Special Circumstances of institute's extension increasing sequence, in the PCR of test kit damping fluid, add the PCR such as methane amide or dimethyl sulfoxide (DMSO) reaction additives to reduce high GC content or the impact of secondary structure on PCR reaction.
Brief description of the drawings
The amplification curve of Fig. 1 visualizingre agent box positive quality control product.Transverse axis represents cycle number (Ct value), and the longitudinal axis represents fluorescence intensity, and ' S ' type curve is saltant type probe color fluorescence curve, and wild-type probe color fluorescence curve is without amplification.
Fig. 2 shows the amplification curve of negative quality control product.Transverse axis represents cycle number (Ct value), and the longitudinal axis represents fluorescence intensity, and ' S ' type curve is wild-type probe color fluorescence curve, and saltant type probe color fluorescence curve is without amplification.This feminine gender quality control product is to be the homozygous plasmid vector of PDS gene IVS7-2 site mutation through order-checking assay.
Fig. 3 shows the amplification curve of negative quality control product.Transverse axis represents cycle number (Ct value), and the longitudinal axis represents fluorescence intensity, and wild-type and saltant type probe color fluorescence curve Ct value are all without amplification.This feminine gender quality control product is salmon sperm dna.
Fig. 4 visualizingre agent box detects the amplification curve of PDS gene IVS7-2A > G homozygous mutation type human peripheral tissue sample.The same Fig. 1 of result.
Fig. 5 visualizingre agent box detects the amplification curve of PDS gene IVS7-2A > G wild-type human peripheral tissue sample.The same Fig. 2 of result.
Fig. 6 visualizingre agent box detects the amplification curve of PDS gene IVS7-2A > G heterozygous human peripheral tissue sample.Transverse axis represents cycle number (Ct value), and the longitudinal axis represents fluorescence intensity, and two ' S ' type curves represent respectively wild-type and saltant type probe color fluorescence curve, and the Ct value of visible 2 curves approaches.
Fig. 7 shows the amplification curve of the human peripheral tissue of DNA extraction failure, the same Fig. 3 of result.Wild-type and saltant type probe color fluorescence curve, all without amplification, illustrate DNA extraction failure.
Embodiment
The following example is intended to illustrate instead of limit the present invention.
Embodiment 1:PDS gene IVS7-2A > G mutation detection kit and use thereof
The composition of test kit
PCR reaction solution 1 is managed (500ul), positive quality control product 1 is managed (20ul), and negative quality control product 1 is managed (20ul), and DNA extraction liquid 1 is managed (3ml), 1 bottle of erythrocyte cracked liquid (15ml).
PCR reaction solution comprises: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U/ml, 0.2uM forward primer (SEQ ID NO:1), 0.2uM reverse primer (SEQ ID NO:2), 0.2uM wild-type fluorescent probe (SEQ ID NO:3), 0.2uM saltant type fluorescent probe (SEQ ID NO:4).
Operation steps:
1) gene extracts: get 100ul EDTA anticoagulation cirumferential blood, add 600ul erythrocyte cracked liquid, room temperature is placed 5-10 minute, mixes during this time for several times centrifugal 5 minutes of 5000rpm, abandon supernatant, then add DNA cleavage liquid 100ul, mix, 99 degree 10 minutes, centrifugal, get supernatant and touch plate as DNA and carry out follow-up work.Also can use business-like DNA extraction test kit to carry out gene extraction, leaching process carries out according to corresponding test kit explanation.
2) PCR detects: get PCR reaction solution 23ul and insert in PCR pipe, add 2ul sample DNA, mix and be placed in quantitative real time PCR Instrument.
Positive quality control: get respectively positive quality control product 2ul and add in PCR reaction solution, remaining same sample operation.
Negative Quality Control: get respectively negative quality control product 2ul and add in PCR reaction solution, remaining same sample operation.
Pcr amplification parameter: 50 DEG C of 2min, 95 DEG C of 2min, then carry out 40 circulations: 95 DEG C of 30sec → 61 DEG C 20sec, preserve result to treat further analysis after reaction finishes.
3) interpretation of result: sample is analyzed according to positive quality control product, negative quality control product fluorescence color curve.If there is the fluorescence curve of 2 different colours, sample is heterozygous.
Embodiment 2:PDS gene IVS7-2A > G mutation detection kit and use thereof
The composition of test kit
PCR reaction solution 1 is managed (500ul), positive quality control product 1 is managed (20ul), and negative quality control product 1 is managed (20ul), and DNA extraction liquid 1 is managed (3ml), 1 bottle of erythrocyte cracked liquid (15ml).
PCR reaction solution comprises: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U/ml, 0.2uM forward primer (SEQ ID NO:1), 0.2uM reverse primer (SEQ ID NO:2), 0.2uM saltant type fluorescent probe (SEQ ID NO:4).
Operation steps is with embodiment 1.
Interpretation of result: sample is analyzed according to positive quality control product fluorescence color curve.If there is the curve identical with positive quality control product fluorescence color, there is PDS gene IVS7-2A > G sudden change in sample, can not distinguish heterozygosis or homozygous mutation.If there is no fluorescence curve, sample is judged to be wild-type.
Claims (7)
1. a method that adopts real-time fluorescence polymerase chain reaction technique (FQ-PCR) to detect PDS gene IVS7-2A > G sudden change, it is characterized in that with PDS gene the 7th intron second-to-last base (IVS7-2A > G) design specificity wild-type probe and saltant type probe, wild-type and saltant type probe are labeled respectively the fluorescence of different colours, simultaneously at PDS gene IVS7-2A > G base both sides design specificity amplification primer, for detecting sample, the fluorescence color that produces in pcr amplification from saltant type probe according to wild-type different come judgement sample whether there is sudden change.
2. a test kit that detects PDS gene IVS7-2A > G sudden change with real time fluorescent PCR method, is characterized in that test kit mainly comprises: PCR reaction solution, negative quality control product, positive quality control product.
3. according to claim 1, method described in 2 and test kit thereof, it is characterized in that, PDS gene IVS7-2A > G alkali yl upstream design forward amplimer, design reverse amplimer in downstream, its length is the nucleotide sequence between 10-30bp, the nucleotide fragments that can to amplify the length that contains PDS gene IVS7-2A > G region be 50-700bp, preferably, forward primer (SEQ ID NO:1) and reverse primer (SEQ ID NO:2) are for can amplify the specificity nucleotide sequence that contains PDS gene IVS7-2 region 60-150bp fragment.
4. according to method and test kit thereof described in claim 1,2, it is characterized in that, PDS gene IVS7-2A > G zone design wild-type probe and saltant type probe, its length is the nucleotide sequence between 10-30bp.Two kinds of probes all cover PDS gene IVS7-2 region, wherein wild-type probe (SEQ ID NO:3) is for PDS gene IVS7-2A > G wild-type site, and saltant type probe (SEQ ID NO:4) is for PDS gene IVS7-2A > G mutational site.
5. according to method and test kit thereof described in claim 1,2,3,4, it is characterized in that, in PCR reaction solution, comprise fluoroscopic examination material.Fluoroscopic examination material is fluorescent probe, its middle probe 5 ' end mark fluorescent generation group, 3 ' end mark fluorescent quenching group.
6. according to method and test kit described in claim 1,2,3,4,5, it is characterized in that, this test kit comprises following content: PCR reaction solution contains 1XPCR Buffer, dNTP, Mg
2+, Taq enzyme, deionized water, forward type primer, reversal primer, wild-type fluorescent probe, saltant type fluorescent probe.
7. according to the test kit described in method and right 6 described in claim 1,2, it is characterized in that blood, saliva, amniocyte or other tissue of target nucleic acid sample to be detected from detected person; The DNA that tissue, blood or the clone that positive quality control product is is PDS gene IVS7-2A > G homozygous mutation type through order-checking assay is extracted or PCR product or the plasmid vector that contains corresponding sequence; Negative quality control product is distilled water, pure water, physiological saline, salmon sperm dna; Negative quality control product also can be for through order-checking assay being tissue, blood or the DNA of clone extraction or PCR product or the plasmid vector that contains corresponding sequence of PDS gene IVS7-2A > G wild-type.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310142823.3A CN104120167A (en) | 2013-04-24 | 2013-04-24 | Method and kit for detecting mutation of PDS gene IVS7-2A>G by using fluorescence PCR technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310142823.3A CN104120167A (en) | 2013-04-24 | 2013-04-24 | Method and kit for detecting mutation of PDS gene IVS7-2A>G by using fluorescence PCR technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104120167A true CN104120167A (en) | 2014-10-29 |
Family
ID=51765797
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310142823.3A Pending CN104120167A (en) | 2013-04-24 | 2013-04-24 | Method and kit for detecting mutation of PDS gene IVS7-2A>G by using fluorescence PCR technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104120167A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711367A (en) * | 2015-04-03 | 2015-06-17 | 济南英盛生物技术有限公司 | Multichannel fluorescent PCR detection kit of delayed deafness gene |
-
2013
- 2013-04-24 CN CN201310142823.3A patent/CN104120167A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 刘明等: "遗传性耳聋家系的SLC26A4 IVS7-2A>G基因突变分析", 《中华耳科学杂志》 * |
| 戴朴等: "1552例中重度感音神经性聋患者SLC26A4基因外显子7和8序列测定及热点突变分析", 《中华医学杂志》 * |
| 袁永一等: "27 个省市聋校学生基于 SLC26A4 基因IVS7-2 A>G突变的全序列分析", 《中华耳科学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711367A (en) * | 2015-04-03 | 2015-06-17 | 济南英盛生物技术有限公司 | Multichannel fluorescent PCR detection kit of delayed deafness gene |
| CN104711367B (en) * | 2015-04-03 | 2016-06-22 | 济南英盛生物技术有限公司 | A kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101684497B (en) | Deafness susceptibility gene screen test kit | |
| Costa et al. | Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene | |
| CN107254531B (en) | Genetic biomarker for auxiliary diagnosis of early colorectal cancer and application thereof | |
| CN103911452A (en) | Chinese population deaf gene screening kit and application thereof | |
| CN102534031A (en) | High-specificity kit for detecting deafness predisposing genes | |
| CN102465174A (en) | Method for detecting GJB2 gene mutation by fluorescence quantitative PCR technology, and kit thereof | |
| CN107227371A (en) | Primer, molecular beacon, kit and its detection method of CYP2C9*3 gene pleiomorphism quick detections | |
| CN111676283B (en) | Application of mitochondrial DNA single nucleotide polymorphism related to occurrence of high altitude pulmonary edema | |
| CN110699446B (en) | SNP marker rs3174298 related to non-syndrome cleft lip and palate diagnosis and application thereof | |
| CN111118138A (en) | Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR | |
| CN104120166A (en) | Method and kit for detecting mutation of PDS gene 2168A>G by using fluorescence PCR technology | |
| CN106834434B (en) | Nucleic acid, kit and method for detecting COX-1, COX-2 and GPIIIa gene polymorphism | |
| EP2130917A1 (en) | Probe for detecting polymorphism in gene involved in immunity, and use thereof | |
| CN105441540A (en) | Non-syndromic deafness gene polymorphism detecting kit and application thereof | |
| CN111560428A (en) | Application of substance for detecting single nucleotide polymorphism of mitochondrial DNA rs3937033 | |
| CN103103259A (en) | Method, kit and primers for determining whether two predetermined loci of nucleic acid sample mutate or not | |
| CN110878351A (en) | Method for detecting MTHFR gene polymorphism by fluorescent quantitative PCR | |
| CN110819709A (en) | Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) | |
| JP2013236627A (en) | Analysis methods for y chromosome and deletion site of sperm forming area on y chromosome | |
| CN116716386A (en) | Detection kit for vitamin C deficiency risk assessment and application method thereof | |
| CN104120167A (en) | Method and kit for detecting mutation of PDS gene IVS7-2A>G by using fluorescence PCR technology | |
| CN104789673B (en) | Applications of the rs1800818 in heating companion's thrombocytopenic syndromes that new bunyavirus causes are detected | |
| KR102333830B1 (en) | Novel mitochondrial genomic markers for the diagnosis of macular degeneration | |
| CN102534030A (en) | Kit for jointly detecting four deafness predisposing genes and application thereof | |
| US10770183B2 (en) | Methods of assessing a risk of developing necrotizing meningoencephalitis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: SHI GUIZHI Free format text: FORMER OWNER: WANG BAOHENG Effective date: 20150211 Owner name: WANG BAOHENG Effective date: 20150211 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Shi Guizhi Inventor after: Wang Baoheng Inventor before: Wang Baoheng Inventor before: Shi Guizhi |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WANG BAOHENG SHI GUIZHI TO: SHI GUIZHI WANG BAOHENG |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20150211 Address after: 250002, room 2, building 9, building 501, provincial court dormitory, sunshine Shun two district, Shizhong District, Ji'nan City, Shandong Applicant after: Shi Guizhi Applicant after: Wang Baoheng Address before: 250002, room 2, building 9, building 501, provincial court dormitory, sunshine Shun two district, Shizhong District, Ji'nan City, Shandong Applicant before: Wang Baoheng |
|
| DD01 | Delivery of document by public notice |
Addressee: Wang Baoheng Document name: the First Notification of an Office Action |
|
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20141029 |