CN106591480A - Multiplex PCR specific primers, kit and method for detecting causative genes of large vestibular aqueduct syndrome based on high throughput sequencing technique - Google Patents

Abstract

The invention belongs to the field of gene detection and relates to multiplex PCR specific primers, kit and method for detecting causative genes of large vestibular aqueduct syndrome based on a high throughput sequencing technique. The invention discloses the multiplex PCR specific primers which are used for detecting causative genes of large vestibular aqueduct syndrome and are shown in formulas of SEQ ID NO: 1 to SEQ ID NO: 86, the kit containing the primers and the detection method for capturing and amplifying the causative gene of large vestibular aqueduct syndrome through a one-step PCR amplification reaction through the primers or kit. The multiplex PCR specific primers, kit and detection method can realizes simultaneous detection of multiple causative genes of large vestibular aqueduct syndrome of multiple samples, effectively improve the detection efficiency and accuracy, reduce a cost and simplify operation processes.

Description

The multiple of large vestibular aqueduct syndrome Disease-causing gene is detected based on high-flux sequence PCR specific primers, test kit and method

Technical field

The invention belongs to field of gene detection, more particularly to a kind of to detect big aquaductus vestibuli based on high throughput sequencing technologies The multiplex PCR specific primer of syndrome Disease-causing gene, test kit and method.

Background technology

Large vestibular aqueduct syndrome (Large Vestibular aqueduct Syndrome, LVAS) is in the seventies A kind of inborn genetic inner ear malformations dysaudia that end is found, is with sensorineural deafness with Large Vestibular Aqueduct (SNHL) a kind of recessive hereditary dysaudia disease being characterized, is clinically mainly shown as that high frequency audition is damaged Phonosensitive nerve deafness based on mistake, shows degree of attaching most importance to deafness more or pole severe is deaf, can be simultaneously with repeatedly A series of clinical symptoms such as the tinnitus of outbreak or dizziness.Morbidity is more in the Childhood, and its premorbid often has flu, fever, wound Etc. the inducement for making increased intracranial pressure.This year, multinomial clinical research showed, non-syndrome deafness infant in FOXI1, KCNJ10, SLC26A4 gene mutation incidence rates are higher, and the patient of the cause of disease is wanted to know about for large vestibular aqueduct syndrome to preliminary suspection, build View carries out FOXI1, KCNJ10, SLC26A4 gene test.Gene test is to large vestibular aqueduct syndrome disorder in screening, prevention And diagnosis has certain effect, the deaf inheritance cause of disease is such as found, is intervened ahead of time, instructed artificial cave or instruct science marriage and childbirth, Reduce the risk for suffering from deafness of future generation.Accomplish " early to find, early prevention, early treatment ".

SLC26A4 (Solute Carrier Family 26Member 4) belongs to No. 4 members in sapiens's Solute Carrier family 26, Protein coding gene on No. 7 chromosomes, size is 57175bp, and 780 aminoacid are encoded altogether, and its biologic activity is Secondary activity sulfate transmembrane transporter activity and chloride ion transmembrane transporter activity, Abroad in Recent Years multiple studies have shown that (aqueduct of vestibule expands or with inner ear malformations nerve deafness and thyroid with Pendred syndromes (PDS) for SLC26A4 gene mutation It is swollen) and large vestibular aqueduct syndrome (LVAS) have close relationship.

FOXI1 (Forkhead Box I1) belongs to jaw transcription factor family, the encoding histone on No. 5 chromosomes Gene, size is 3829bp, and 378 aminoacid are encoded altogether, and its biologic activity position is transcription factor activity and sequence-specific DNA binding activity, the development to cochlea and vestibule has vital effect.And FOXI1 gene mutation then may be by affecting In ear chamber phenotype normal function is affecting normal good hearing.

KCNJ10 (Potassium Voltage-Gated Channel Subfamily J Member 10) belongs to introversive Rectification type potassium channel family, the protein coding gene on No. 1 chromosome, gene size is 71421bp, and 379 are encoded altogether Aminoacid, its passage relational approach includes inward rectification K+ passages and potassium channel, and coded protein also can be with another kind of potassium Channel protein forms heterodimer, is responsible for the potassium cushioning effect of brain mesoglia cells, and for cell potassium ion input is provided Passage.And KCNJ10 gene mutation then may be by affecting potassium channel normal function to affect audition to develop.

At present, detect that large vestibular aqueduct syndrome common methods have Sanger methods, gene chips, quantitative fluorescent PCR Method, multicolor fluorescence solubility curve analytic process, high-flux sequence method etc..The sequencing of Sanger sequencing every time can only be to sample A certain section of region is sequenced, and detection efficiency is low, high cost, and sensitivity low (20%).Gene chips, quantitative fluorescent PCR Method detects large vestibular aqueduct syndrome Disease-causing gene, and the method can only be directed to the site design inspection of or several known mutations Probing pin forms detection chip, therefore is not used to detect unknown mutation site, and the result of detection is also just not enough comprehensively, accurately. Multicolor fluorescence solubility curve analytic process, fluorescent dye used is general double-stranded DNA intercalator, to primer dimer and Fei Te Different amplified production None- identified, easily causes false positive.The survey that high-flux sequence method is mainly provided by sequencing instrument provider The supporting library construction Kit of sequence instrument, is assessed by supply company's Quality Control, be there is test kit delivery date length, test kit specification and is fixed, tries Test motility and not enough wait not enough.Some reagent manufacturers carry out building storehouse according to the experimental technique for sharing report in document, but are completed Experiment effect it is uneven, sequencing quality is not high.

The content of the invention

In consideration of it, being necessary to provide a kind of comprehensive based on the big aquaductus vestibuli of high-flux sequence detection of platform for the problems referred to above The multiplex PCR specific primer of simulator sickness Disease-causing gene, test kit and experimental technique, can detect simultaneously multiple samples it is multiple it is big before Front yard aqueduct syndrome Disease-causing gene, effectively improves detection efficiency, accuracy rate, while reduces cost, simplified operating procedure etc..

To reach above-mentioned purpose, the present invention is employed the following technical solutions：

It is a kind of to detect that the multiplex PCR specificity of large vestibular aqueduct syndrome Disease-causing gene draws based on high throughput sequencing technologies Thing, including 43 pairs of specific primers, the specific primer its nucleotide sequence such as SEQ ID NO：1 to SEQ ID NO：86 institutes Show.

Further, the specific primer is the primer through modifying.

Further, the method for modifying of the specific primer is thio-modification, Brdurd is modified, 5 ' reverse dT are repaiied One or more in decorations.

Further, the Tm values of the specific primer are 58-62 DEG C.

A kind of test kit that large vestibular aqueduct syndrome Disease-causing gene is detected based on high throughput sequencing technologies, including it is above-mentioned At least two pairs in specific primer.

Further, the test kit also includes universal primer, enzymatic mixture, quality-control product, nuclease free water；The enzyme is mixed Compound includes：High-fidelity DNA polymerase, PCR Buffer, dNTP mixture etc..

Further, quality-control product behaviour normal gene group DNA.

Further, the end of specific primer 3 ' and 5 ' end difference labelling Tag1 and Tag2.

Further, the end Tag1 sequences of specific primer 3 ' and target area complete complementary or partial complementarity；Specifically Property end Tag2 and the universal primer complete complementary of primer 5 ' or partial complementarity.

Further, the universal primer is used to expand special primer amplification target area again, and difference is treated The target area amplified production of test sample product is marked.

Further, the universal primer 3 ' end Tag3 and special primer Tag2 complete complementaries or partial complementarity, length is 50-70bp。

The further specific primer or test kit are applied to the detection of large vestibular aqueduct syndrome Disease-causing gene.

Based on the method that high throughput sequencing technologies detect large vestibular aqueduct syndrome Disease-causing gene, comprise the following steps：

S1：Nucleic acid extraction is carried out to sample using paramagnetic particle method, the measure of concentration and purity is then carried out, is determined qualified laggard The quantitative dilution of row, it is standby；

S2：Carried out once with the target area of the nucleic acid of the specific primer or the test kit to being extracted in S1 PCR is expanded, and collects amplified production standby；

S3：Nucleic acid purification is carried out to amplified production in S2 using paramagnetic particle method, the measure of concentration and purity is carried out, sample mixing is fixed Amount, upper machine sequencing.

Further, the PCR reaction systems are：

The enzymatic mixture includes：High-fidelity DNA polymerase, PCR Buffer, dNTP mixture etc..

Further, the PCR response procedures are：

Further, the large vestibular aqueduct syndrome Disease-causing gene includes FOXI1 (primer sequence SEQ ID NO:1- SEQ ID NO:12), KCNJ10 (primer sequence SEQ ID NO:13-SEQ ID NO:40), SLC26A4 (primer sequence SEQ ID NO:41-SEQ ID NO:86)。

Further, the target area of Disease-causing gene described in step (2), including but not limited to FOXI1, KCNJ10, The outside regulating and controlling sequence of internal sequence, gene on SLC26A4 genes.The gene internal sequence, including but not limited to gene Include subregion, exon region, simultaneously containing the region of intron and exon.

Further, the detection object of the detection method includes in vitro whole blood, mouth desquamated cells and tissue sample Deng；The tissue sample includes paraffin-embedded tissue, flesh tissue and frozen section etc..

Ion Torrent/ of the above-mentioned high-flux sequence platform including but not limited to Illumina HiSeq/MiSeq, Life The microarray datasets such as Proton platforms, the BGISEQ-500/50 of Hua Da gene.

Beneficial effect of the present invention：

The primer that the present invention is provided is for large vestibular aqueduct syndrome Disease-causing gene FOXI1, KCNJ10, SLC26A4 Full exon design primer, wide coverage, detection site are more.Each pair primer amplified target area size is 450-550bp, all primer Tm Unitings are 58-62 DEG C.

Special sex modification, such as thio-modification, deoxidation urine are introduced during the multiplex PCR specific primer design that the present invention is provided The method of modifying such as pyrimidine modification, 5 ' reverse dT modifications, the primer after modification is difficult by nuclease degradation, it is ensured that PCR amplifying specifics Ensure its amplification efficiency while property；And with relatively uniform Tm values, good specificity, steady is shown in multiplex amplification Qualitative and homogeneity, while there is no non-specific amplification, the method saves the work of many wheel primer screenings.Specifically, it is thio to repair The derivant that the oxygen atom of the primer sequence oligonucleotide chain upper band double bond after decorations is replaced by sulphur atom, can resist nuclease Degraded, strengthens primer stability；Each deoxythymidine is taken off in primer oligonucleotide sequence after Brdurd modification Oxygen uracil is substituted, and double-strand solution temperature can be made to increase by 1.7 DEG C, so as to increase the stability of double-strand；After 5 ' reverse dT modifications The end of primer oligonucleotide sequence 5 ' contain reverse dT, so as to form crosslinking, the crosslinking of formation can suppress in archaeal dna polymerase The Degradation of the exonuclease during extension.

Independent research of the present invention it is higher for large vestibular aqueduct syndrome sickness rate based on high-flux sequence platform The multiplex PCR specific primer of Disease-causing gene, test kit and detection method, with a pcr amplification reaction capture and can expand Increase the multiple large vestibular aqueduct syndrome Disease-causing genes of sample, including the heritability such as point mutation, short-movie section insertion and deletion is dashed forward Become, effectively improve detection efficiency, accuracy rate, while reduces cost, the operating procedure that simplifies, flexible offer kit package specification Deng.The present invention breaks through the restriction of traditional sensing techniques using high flux gene sequencing technology, improves detection sensitivity；Simultaneously can also Detection includes the variation situation in known mutations and unknown mutation site, obtains comprehensive and accurate testing result.By to big vestibule The Disease-causing gene of aqueduct syndrome and its mutational site are sequenced, and specific gene sequence information is obtained, and then are assessed The genetic risk of large vestibular aqueduct syndrome, accomplishes early prevention, early control, reduces disease occurrence probability, improves health of people Level.

The present invention eliminates the centrifugation pillar purification that tradition is built in storehouse by the way of magnetic beads for purifying nucleic acid, simple to operate； Secondly, the present invention only needs to a wheel PCR for the primer of high-flux sequence Platform Designing, by choosing thermograde, time ladder Degree, period optimize PCR response procedures through a large amount of orthogonal experiments, and the fusion of amplification twice that sample is built into storehouse is anti-in a PCR System is answered to carry out, and the inventive method primer directly captures target area in target gene chain and is expanded, it is not necessary to Target gene is carried out to block the process such as modification, detection time and cost is saved.

Description of the drawings

Fig. 1 is the method flow diagram for detecting large vestibular aqueduct syndrome Disease-causing gene.

Fig. 2 builds the schematic diagram of storehouse PCR amplifications for target gene.

Fig. 3 is primer specificity the result figure；Wherein, M：Marker；N：Negative control；P：Positive control；1-3：1-3 The checking of number whole blood sample；4-6：4-6 mouth desquamated cells sample is verified；7-9：7-9 paraffin organizations sample is verified.

Fig. 4 is primer sensitivity the result figure；Wherein, M：Marker；N：Negative control；P：Positive control；1-3:No. 1 Three kinds of template concentrations sensitivity checkings of whole blood sample；4-6：The three kinds of template concentrations sensitivity checkings of No. 4 mouth desquamated cells samples； 7-9：The three kinds of template concentrations sensitivity checkings of No. 7 paraffin organization samples.

Fig. 5 is primer repeatability the result；Wherein, M：Marker；N：Negative control；P：Positive control；1-3：Experiment Member A detects No. 2 whole blood samples, No. 5 mouth desquamated cells samples, No. 8 paraffin organization sample the results；4-6：Laboratory technician B is examined Survey No. 2 whole blood samples, No. 5 mouth desquamated cells samples, No. 8 paraffin organization sample the results.

Specific embodiment

In order to problem solved by the invention, the technical scheme for being adopted and the effect for being reached is better described, now tie Close specific embodiment and related data is expanded on further.It should be noted that present invention is including but not limited to following enforcement Example and combinations thereof embodiment.

A kind of multiplex PCR that large vestibular aqueduct syndrome Disease-causing gene is detected based on high throughput sequencing technologies of embodiment 1 Specific primer, test kit and method

1st, the design of primer

It is described that University of California are selected to large vestibular aqueduct syndrome Disease-causing gene sequence Santa Cruz (UCSC) data base, from Primer3 primer-design softwares the full exon design of primers of Disease-causing gene is carried out, if Meter scope contains all common and uncommon mutantional hotspot in the gene.

The design of primers carried out to the full exon of large vestibular aqueduct syndrome Disease-causing gene totally 43 pairs, each pair is special Property primer amplification target area size be 450-550bp, wide coverage, detection site are more；Introduce when special primer is designed special Opposite sex modification, the primer after modification is difficult by nuclease degradation, and with relatively uniform Tm values, Uniting is 60 DEG C or so (±2℃)；The primer shows good specificity, stability and homogeneity in multiplex amplification, while there is no non-spy Different amplification.Specifically, the primer specific method of modifying is in thio-modification, Brdurd modification, 5 ' reverse dT modifications One or more.The oxygen atom of the primer sequence oligonucleotide chain upper band double bond after the thio-modification is by spreading out that sulphur atom replaces Biology, can resist the degraded of nuclease, strengthen primer stability；Primer tasteless nucleotide sequence after the Brdurd modification Each deoxythymidine is deoxidized uracil replacement in row, double-strand solution temperature can be made to improve 1.7 DEG C, so as to increase double-strand Stability；Reverse dT is contained in the end of primer oligonucleotide sequence 5 ' after described 5 ' reverse dT modifications, so as to form crosslinking, shape Into crosslinking can suppress archaeal dna polymerase extend during exonuclease Degradation.

The sequencing primer of the present invention is, through Mdification primer, can to better assure that PCR specific amplifications and its amplification efficiency.

2nd, large vestibular aqueduct syndrome Disease-causing gene test kit is detected

The test kit of present invention detection large vestibular aqueduct syndrome Disease-causing gene method, including：

Large vestibular aqueduct syndrome Disease-causing gene specific primer：Multiple target areas in for expanding testing sample, The full exon region of amplification scope at least coverage goal gene；

Universal primer：For in library construction process, carrying out again to the amplified production that special primer expands target area Secondary amplification, is marked to the sequencing library of different testing samples, and then distinguishes different samples；

PCR reactant liquors：Including enzymatic mixture, nuclease free water；The enzymatic mixture includes that high-fidelity DNA polymerase (is used Measure as 1.25U/ reactions), PCR Buffer (consumption be 1 ×), dNTP mixture (consumption is 200 μM/kind/reaction)；

Quality-control product：People's normal gene group DNA, from normal person's whole blood sample, present invention test quality-control product used comes Come from commercially available.

3rd, the method for detecting large vestibular aqueduct syndrome Disease-causing gene based on high throughput sequencing technologies, including following step Suddenly：

S1：Using nanoscale, superparamagnetic carboxyl magnetic bead under different salinity and hydrophobic environment with the reversible suction of nucleic acid molecules Attached principle, Specific adsorption nucleic acid molecules, by subsequently washing and elution step, obtain highly purified sample of nucleic acid, use Qubit or NanoDrop carries out the measure of concentration and purity, according to the sample of nucleic acid concentration results for determining, using 10mM Tris Sample of nucleic acid is diluted to into 50-200ng/ μ L, preferred 50-100ng/ μ L build the initial concentration in storehouse as amplification.

Using 1% agarose gel electrophoresiies 40min, the quality of nucleic acid after dilution is estimated, according to agarose gel The homogeneity of electrophoresis result and integrity judge the quality for extracting nucleic acid, and nucleic acid integrity is good or generation slight degradation does not affect to build Storehouse effect.Enter group after the detection of the nucleic acid concentration that extracted, purity testing and agarose gel electrophoresiies is qualified and be marked respectively. The qualified standard of the sample is nucleic acid concentration ＞ 50ng/ μ L, OD260/OD280=1.7-2.0,1% agarose gel electrophoresiies Detection sample band is complete, homogeneous, without substantially hangover.

S2：The Disease-causing gene target area in testing sample in S1 is carried out with the specific primer or test kit of the present invention PCR amplification, collects amplified production, obtains building storehouse product；The amplified production fragment length is special primer amplified fragments With universal primer sum；

Pcr amplification reaction system is：The μ L of special primer mixture 2；The μ L of universal primer 2；Template/quality-control product 100ng；Enzyme is mixed The μ L of compound 12.5；Volume is complemented to 25 μ L by nuclease free water.

PCR response procedures are：

When large vestibular aqueduct syndrome Disease-causing gene to be measured has multiple, the big vestibule water guide of difference of same testing sample The amplification of pipe syndrome Disease-causing gene can be carried out simultaneously or part is while carry out.In specific experimentation, can be as needed Carried out from any of the above-described kind of scheme.If expanded respectively, guarantee is needed for building the amplified production of sequencing library Amount is consistent.When testing sample has it is multiple when, the amplification of the large vestibular aqueduct syndrome Disease-causing gene of different samples is necessary Carry out respectively.

S3：Respectively purification is carried out to the storehouse product of building of each sample using purification magnetic bead.Remove remaining primer in PCR primer And dNTP etc., reclaim amplified production；Carry out concentration, purity testing and 1% agarose gel electrophoresiies detection sample quality, the sample This qualified standard is nucleic acid concentration ＞ 2ng/ μ L, OD260/OD280=1.7-2.0,1% agarose gel electrophoresiies detection samples Band is complete, homogeneous, without substantially hangover.All qualified nucleic acid samples (in principle less than 96) concentration are diluted to respectively 2nmol/L, and equal-volume sample mixing, machine sequencing in unification.

Controlled trial：

Positive control reaction system includes：The μ L of special primer mixture 2；The μ L of universal primer 2；The μ L of enzymatic mixture 12.5；Matter Control product 100ng；Volume is complemented to 25 μ L by nuclease free water.Just whether positive control be used to check the amplification ability of reaction system Whether normal and sample is abnormal.

Negative control reaction system includes：The μ L of special primer mixture 2；The μ L of universal primer 2；The μ L of enzymatic mixture 12.5；Nothing Volume is complemented to 25 μ L by nuclease water.Negative control is used to check that reaction system pollutes with the presence or absence of nucleic acid substances.

The response procedures of positive control and negative control with step S2 amplified reaction program.

The primer specificity of embodiment 2 is verified

Using S1 methods in embodiment 1 from whole blood sample (numbering：1-3), mouth desquamated cells sample (numbering：4-6), stone Wax tissue sample (numbering：Nucleic acid is extracted in 7-9), after carrying out concentration, purity testing, taking qualified samples will using 10mM Tris Every diluted sample detects every sample quality (same embodiment of criterion of acceptability to 100ng/ μ L using 1% agarose gel electrophoresiies S1 steps in 1), it is qualified after enter group and be marked respectively.Above-mentioned 9 qualified samples are carried out using S2 methods in embodiment 1 Amplification, applied sample amount is each 1 μ L, carries out 1% agarose gel electrophoresiies detection (the same embodiment of criterion of acceptability after amplification after product purification S3 steps in 1).9 samples are expanded using large vestibular aqueduct syndrome Disease-causing gene special primer and detection method is examined Survey.Controlled trial is with embodiment one, and detection method is identical with sample testing method.Testing result is shown in Fig. 3.

Testing result represents that negative control, without any non-specific band, illustrates that reaction system is without dirt in addition to residual primers Dye；Positive control band becomes clear, and size is correct, illustrates that the amplification ability of reaction system is normal.9 sample amplified productions of detection 600bp is concentrated mainly on, without other non-specific bands, is consistent with desired design, and primer dimer content is few, and primer Dimer size and amplification target fragment obvious difference.It can thus be seen that the pcr amplification primer thing and detection method energy of the present invention The restriction that traditional method produces a large amount of primer dimers is broken through, primer specificity is enhanced.

The primer detection sensitivity of embodiment 3 is verified

Using S1 methods in embodiment 1 from the qualified whole blood sample of quality inspection (numbering：1), mouth desquamated cells sample (is compiled Number：4), paraffin organization sample (numbering：7) extracting sample nucleic carries out primer detection sensitivity checking.Every sample initial concentration For 100ng/ μ L, according to 5 times, 10 times, 20 times of Concentraton gradient dilutions, after dilution every sample concentration be respectively 20ng/ μ L, 10ng/ μ L and 5ng/ μ L, and sample ID and concentration markers are carried out respectively.It is qualified to above-mentioned 9 using S2 methods in embodiment 1 Diluted sample is expanded, and applied sample amount is each 1 μ L, and 9 samples use large vestibular aqueduct syndrome Disease-causing gene special primer Amplification and detection method are detected.Controlled trial is with embodiment one, and detection method is identical with sample testing method.Testing result See Fig. 4.

Testing result represents that negative control, without any non-specific band, illustrates that reaction system is without dirt in addition to residual primers Dye；Positive control band becomes clear, and size is correct, illustrates that the amplification ability of reaction system is normal.9 sample amplified productions of detection 600bp is concentrated mainly on, without other non-specific bands, is consistent with desired design, while primer dimer content is few, and drawn Thing dimer size and amplification target fragment obvious difference.Meanwhile, even if template content as little as 5ng, still being capable of Successful amplification Go out target stripe, it can thus be seen that the pcr amplification primer thing and detection method of the present invention can break through the restriction of traditional method, carry High detection sensitivity.

The repeatability checking of the primer detection of embodiment 4

Using S1 methods in embodiment 1 from the qualified whole blood sample of quality inspection (numbering：2), mouth desquamated cells sample (is compiled Number：5), paraffin organization sample (numbering：8) extracting sample nucleic carries out primer detection repeatability checking.Every sample by 2 not Same operator S2 methods in 2 different lab platforms are according to embodiment 1 are expanded to above-mentioned 3 samples, loading Measure as 1 μ L, 3 samples are using large vestibular aqueduct syndrome Disease-causing gene special primer by different operating person in different experiments Room expands and detection method is detected.Controlled trial is with embodiment one, and detection method is identical with sample testing method.Detection knot Fruit sees Fig. 5.

Testing result represents that negative control, without any non-specific band, illustrates that reaction system is without dirt in addition to residual primers Dye；Positive control band becomes clear, and size is correct, illustrates that the amplification ability of reaction system is normal.Totally 6 expansions of 3 samples of detection Volume increase thing is concentrated mainly on 600bp, without other non-specific bands, is consistent with desired design, while primer dimer content pole It is few, and primer dimer size and amplification target fragment obvious difference.Meanwhile, different operators and different experiment porch examine Survey result zero difference.It can thus be seen that the pcr amplification primer thing and detection method of the present invention are reproducible.

Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but and Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art For, without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the guarantor of the present invention Shield scope.Therefore, the protection domain of patent of the present invention should be defined by claims.

<110>Xiangya Hospital, Central-South China Univ.；Guangzhou Qi Hui bio tech ltd

<120>Multiplex PCR specific primer, the examination of large vestibular aqueduct syndrome Disease-causing gene are detected based on high-flux sequence Agent box and

Method

<160> 86

<170> PatentIn version 3.3

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tgggtaggta ggtctatgag ttcag 25

<210> 41

<211> 21

<212> DNA

<213>Artificial sequence

<400> 41

cctcgcaacc cttgagatta g 21

<210> 42

<211> 20

<212> DNA

<213>Artificial sequence

<400> 42

ttaagagtcc catccgcaac 20

<210> 43

<211> 20

<212> DNA

<213>Artificial sequence

<400> 43

ctccgatcgt cctcgcttac 20

<210> 44

<211> 19

<212> DNA

<213>Artificial sequence

<400> 44

ccgctccgct tctctctac 19

<210> 45

<211> 21

<212> DNA

<213>Artificial sequence

<400> 45

tgcaaattgg ttgtgactga g 21

<210> 46

<211> 20

<212> DNA

<213>Artificial sequence

<400> 46

cagacctatg gtagctgggg 20

<210> 47

<211> 24

<212> DNA

<213>Artificial sequence

<400> 47

cattgtaagt tgaggacttt ctgc 24

<210> 48

<211> 23

<212> DNA

<213>Artificial sequence

<400> 48

gccaaaacac tttaaacatg agc 23

<210> 49

<211> 21

<212> DNA

<213>Artificial sequence

<400> 49

tgcagacaca ttgaacattt g 21

<210> 50

<211> 23

<212> DNA

<213>Artificial sequence

<400> 50

ttccaggaaa ttactttgtt ttg 23

<210> 51

<211> 26

<212> DNA

<213>Artificial sequence

<400> 51

aagcttgatg taatatttcc agagag 26

<210> 52

<211> 20

<212> DNA

<213>Artificial sequence

<400> 52

aatgaacagt gacccatccc 20

<210> 53

<211> 19

<212> DNA

<213>Artificial sequence

<400> 53

tgtgctcgtg tgcgtgtag 19

<210> 54

<211> 24

<212> DNA

<213>Artificial sequence

<400> 54

gaaggagtat cagtgaaatg aagc 24

<210> 55

<211> 20

<212> DNA

<213>Artificial sequence

<400> 55

tagcatttga tgagatgggg 20

<210> 56

<211> 21

<212> DNA

<213>Artificial sequence

<400> 56

gctgacacca aaatcttttc c 21

<210> 57

<211> 25

<212> DNA

<213>Artificial sequence

<400> 57

ggtcttgcaa agattcaatt tgtag 25

<210> 58

<211> 20

<212> DNA

<213>Artificial sequence

<400> 58

tgccattcct cgacttgttc 20

<210> 59

<211> 20

<212> DNA

<213>Artificial sequence

<400> 59

caagggagaa ggacgaatcc 20

<210> 60

<211> 24

<212> DNA

<213>Artificial sequence

<400> 60

caggaagcat ataagaacca aatc 24

<210> 61

<211> 20

<212> DNA

<213>Artificial sequence

<400> 61

tgggcaatag agtgtgaccc 20

<210> 62

<211> 21

<212> DNA

<213>Artificial sequence

<400> 62

aacgaaagaa agtggcttca c 21

<210> 63

<211> 20

<212> DNA

<213>Artificial sequence

<400> 63

ttccaaaata cggctgttcc 20

<210> 64

<211> 22

<212> DNA

<213>Artificial sequence

<400> 64

tcaggctagt aaacccaagt cc 22

<210> 65

<211> 23

<212> DNA

<213>Artificial sequence

<400> 65

tccttgctaa gtagccagaa atg 23

<210> 66

<211> 20

<212> DNA

<213>Artificial sequence

<400> 66

tattcctgat tggaccccag 20

<210> 67

<211> 21

<212> DNA

<213>Artificial sequence

<400> 67

ttaggtgcca ggcattttaa g 21

<210> 68

<211> 20

<212> DNA

<213>Artificial sequence

<400> 68

ctgctctcat cagggaaagg 20

<210> 69

<211> 25

<212> DNA

<213>Artificial sequence

<400> 69

ttggataatt tgatatgaat ggttg 25

<210> 70

<211> 20

<212> DNA

<213>Artificial sequence

<400> 70

gccctgttgc aatactggac 20

<210> 71

<211> 23

<212> DNA

<213>Artificial sequence

<400> 71

tgaattatgg gcagataagg ttg 23

<210> 72

<211> 22

<212> DNA

<213>Artificial sequence

<400> 72

gcttacggga aagtcttaca gg 22

<210> 73

<211> 20

<212> DNA

<213>Artificial sequence

<400> 73

agtgagcaat gatgccactg 20

<210> 74

<211> 24

<212> DNA

<213>Artificial sequence

<400> 74

tgaggctcca tgaagttata tagg 24

<210> 75

<211> 20

<212> DNA

<213>Artificial sequence

<400> 75

tttcagtgga gcatcaggtg 20

<210> 76

<211> 20

<212> DNA

<213>Artificial sequence

<400> 76

ggggaattat gttccctgac 20

<210> 77

<211> 24

<212> DNA

<213>Artificial sequence

<400> 77

gcagacttaa ggagaattca gttg 24

<210> 78

<211> 19

<212> DNA

<213>Artificial sequence

<400> 78

atggtgatca gtgcagctc 19

<210> 79

<211> 20

<212> DNA

<213>Artificial sequence

<400> 79

cctctgtggt caagcgagtc 20

<210> 80

<211> 20

<212> DNA

<213>Artificial sequence

<400> 80

acctatacat gcctccatgc 20

<210> 81

<211> 22

<212> DNA

<213>Artificial sequence

<400> 81

cctaaaagct acagaaccag gc 22

<210> 82

<211> 20

<212> DNA

<213>Artificial sequence

<400> 82

aagtccccaa gatcaaagcc 20

<210> 83

<211> 20

<212> DNA

<213>Artificial sequence

<400> 83

tgcatggagg catgtatagg 20

<210> 84

<211> 18

<212> DNA

<213>Artificial sequence

<400> 84

cgcctggcct acattcac 18

<210> 85

<211> 24

<212> DNA

<213>Artificial sequence

<400> 85

tgaaatacaa ccagaaacaa tgtg 24

<210> 86

<211> 21

<212> DNA

<213>Artificial sequence

<400> 86

cgtaactttg gcatttcctt c 21

Claims (10)

1. it is a kind of based on high-flux sequence detect large vestibular aqueduct syndrome Disease-causing gene multiplex PCR specific primer, its It is characterised by, the multiplex PCR specific primer includes 43 pairs of specific primers, its nucleotide sequence such as SEQ ID NO：1 to SEQ ID NO：Shown in 86.

2. specific primer according to claim 1, it is characterised in that the primer is the primer through modifying.

3. specific primer according to claim 2, it is characterised in that the method for modifying of the specific primer is thio One or more in modification, Brdurd modification, 5 ' reverse dT modifications.

4. specific primer according to claim 1, it is characterised in that the Tm values of the specific primer are 58-62 DEG C.

5. the specific primer according to any one of claim 1-4, it is characterised in that the primer is applied to big vestibule and leads Aqueduct syndrome Disease-causing gene is detected.

6. it is a kind of based on high-flux sequence detect large vestibular aqueduct syndrome Disease-causing gene test kit, it is characterised in that bag Include at least two pairs in the specific primer described in any one of the claims 1-4.

7. test kit according to claim 6, it is characterised in that the test kit also include universal primer, enzymatic mixture, Quality-control product, nuclease free water.

8. a kind of method for detecting large vestibular aqueduct syndrome Disease-causing gene based on high-flux sequence, comprises the following steps：

S1：Nucleic acid extraction is carried out to sample using paramagnetic particle method, the measure of concentration and purity is then carried out, it is fixed to carry out after measure is qualified Amount dilution, it is standby；

S2：With the test kit described in the arbitrary described specific primer of claim 1-4 or claim 6 or 7 to being carried in S1 The target area of the nucleic acid for taking carries out a PCR amplification, collects amplified production standby；

S3：Nucleic acid purification is carried out to amplified production in S2 using paramagnetic particle method, and carry out the measure of concentration and purity, sample mixing, quantitatively, Upper machine sequencing.

9. detection method according to claim 8, it is characterised in that the PCR reaction systems are：

10. detection method according to claim 8, it is characterised in that the PCR response procedures are：