CN104711367B - A kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit - Google Patents
A kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit Download PDFInfo
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Abstract
The invention discloses a kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, for on tardy deaf-related gene PDS:IVS7-2A > G, 2168A > G, 1174A > T, 1229C > T, 1226G > A, 1975G > C, 2027T > A the design 4 of totally seven sites to specific primer and 7 TaqMan probe, using mankind house-keeping gene β-Actin as internal reference, design 1 is to specific primer and probe, effectiveness with monitor sample template, reaction system, avoiding the occurrence of false negative, setting negative, positive quality control simultaneously ensures testing result accuracy。The present invention achieves application multichannel fluorescence PCR method first and detects seven mutational sites that Delayed onset is deaf in same reaction tube simultaneously, the method is easy and simple to handle quickly, accurate, high flux, cost are low, cross-contamination is avoided in stopped pipe operation, make that deaf examination purposiveness is strong, examination scope is wide, effectively prevention Delayed onset deafness occurs, it is easy to promote and application。
Description
Technical field
The present invention relates to field of gene detection, be specifically related to a kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit。
Background technology
Deafness is one of clinical common disabled disease, can deafness be divided three classes according to the affected character of auditory system, reason and diseased region clinically: transmission deafness (external ear or Middle Ear Diseases), phonosensitive nerve deafness (internal ear pathological changes), mixed deafness。Research data display phonosensitive nerve deafness, mixed deafness and heredity are closely related, heredity the deafness caused accounts for more than the 60% of deafness。
Delayed onset deafness patient, because carrying PDS gene, in developmental process under the stimulation of risk factor (head trauma, strenuous exercise, flu etc.), can develop into severe deafness or complete deafness。The mode of inheritance of Delayed onset deafness is autosomal recessive inheritance, AR, cause that the deaf gene suddenlyd change is PDS gene, IVS7-2A > G, 2168A > G, 1174A > T, 1229C > T, 1226G > A, 1975G > C, 2027T > A is the mutational site that PDS gene is common, accounts for about the 70% of all sudden changes of PDS gene。In normal population, carrying rate is 1-2%, and in deaf crowd, recall rate is 10-15%。
PDS common causes one of deaf gene, its sudden change it is modal inner ear malformations that the aqueduct of vestibule caused expands。PDS gene is positioned at No. 22 districts of district-31 of No. 7 chromosome long arm, sudden change is caused the high sepage of the endolymphatic sac aquaductus vestibuli through expanding and is returned and vestibule by the backflow substrate of membranous cochlea of ductus reuniens, thus damaging sensory nerve epithelial cell, when slight cranial traumas or any reason causing increased intracranial pressure make the intracranial pressure fluctuation EVA through expanding reach internal ear, membranous labyrinth is broken and is made the mixing of interior perilympha produce prominent deaf, tinnitus, dizziness。Clinical main manifestations is large vestibular aqueduct syndrome, is commonly called as " spank causes deaf "。By PDS gene common mutations site is detected, can avoid and effectively delay the generation of deafness。
Along with the development of technique of gene detection and the concern to audition health, the method used by test kit detecting deafness in the market mainly has gene chips, solubility curve method, ARMS-PCR method, RFLP method。Chip method, it is generally required to steps such as extraction, pcr amplification, hybridization, washing, scannings, operates relatively complicated and time consumption long, requires higher to operator and experimental site。The temperature control of instrument is required significantly high by solubility curve method, it is desirable to instrument has high-resolution solubility curve and analyzes (HRM) function, and the ABI line fluorescent quantitative PCR apparatus that we commonly use generally lacks this function。ARMS-PCR method and RFLP method need PCR primer is carried out electrophoretic analysis, are unsuitable for clinical practice。
Chinese patent " method of Fluorescence PCR assay detection PDS gene IVS7-2A > G sudden change and test kit thereof " (CN104120167A) discloses a kind of method adopting fluorescent quantitative PCR technique detection deaf gene PDS gene IVS7-2A > G sudden change and test kit, but one PCR reaction tube of this test kit is only capable of a mutational site on detection deaf gene PDS, other sites such as the 2168A with higher mutation rate > G, 1229C > T, 1174A > T cannot be detected, thus reducing Delayed onset deafness examination coverage rate。
Summary of the invention
For the deficiency that above-mentioned prior art exists, it is an object of the invention to provide a kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, this test kit can exclusively detect seven sites common in Delayed onset deafness, these seven sites can be detected simultaneously in one PCR reaction tube, specify the classification in high mutation rate site, determine low mutation rate site scope, and it is simple and efficient to handle, the objective controllability of result is strong, efficiency is high, cost is low, stopped pipe operation is anti-pollution, it is suitable for multichannel fluorescent PCR instrument, it it is the strong product carrying out Delayed onset deafness detection clinically。
For achieving the above object, the present invention adopts following technical proposals:
A kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, IVS7-2A with PDS gene > G, 2168A > G, 1174A > T, 1229C > T, 1226G > A, 1975G > C, 2027T > A sports detection object, design specific primer and probe, described specific primer includes for expanding IVS7-2A > primer 1 of G, its sequence is such as shown in SEQ ID NO:1 and SEQIDNO:2；For expanding 2168A > primer 2 of G, its sequence is such as shown in SEQ ID NO:3 and SEQIDNO:4；For expanding 1174A > T, 1229C > T, 1226G > primer 3 of A, its sequence is such as shown in SEQ ID NO:5 and SEQIDNO:6；For expanding 1975G > C, 2027T > primer 4 of A, its sequence is such as shown in SEQ ID NO:7 and SEQIDNO:8；Described probe is whether the IVS7-2 of specific detection PDS gene, 2168,1174,1229,1226,1975,2027 site contains sudden change, and its probe sequence is respectively as shown in SEQ ID NO:9 to SEQIDNO:15。
Concrete, owing to IVS7-2 and 2168 are positioned on the exon that PDS gene is different, distant, therefore separately design primer, IVS7-2 site primer (primer 1) position is selected in the conserved region of IVS7-2 site two flank, and expanding fragment length is 142bp；2168 site primer (primer 2) positions are selected in the conserved region of 2168 site two flanks, and expanding fragment length is 117bp；1174, closely located on PDS gene of 1229,1,226 3 sites, therefore share pair of primers (primer 3), to avoid primer too much to interfere, forward primer is selected in 1174 site upstream conserved region, reverse primer is selected in 1229 sites downstream conserved region, and expanding fragment length is 250bp；1975, closely located on PDS gene of 2,027 two sites, therefore share pair of primers (primer 4), forward primer is selected in 1975 site upstream conserved region, and reverse primer is selected in 2027 sites downstream conserved region, and expanding fragment length is 224bp。Described primer sequence is as follows:
Primer 1F:5'-AACCAATGGAGTTTTTAACAT-3'(SEQIDNO:1)
Primer 1R:5'-TAAGAGGAACACCACACTCAC-3'(SEQIDNO:2)
Primer 2 F:5'-CTGGAGCAATGCGGGTTC-3'(SEQIDNO:3)
Primer 2 R:5'-GGAACCTTGACCCTCTTG-3'(SEQIDNO:4)
Primer 3F:5'-AAATACTCAGCGAAGGTCTTGC-3'(SEQIDNO:5)
Primer 3R:5'-CGAGCCTTCCTCTGTTGC-3'(SEQIDNO:6)
Primer 4F:5'-TCCCAACCAAGGAAATA-3'(SEQIDNO:7)
Primer 4R:5'-GCAATACTGGACAACCC-3'(SEQIDNO:8)
It should be understood that the quality of primer can directly influence specificity and the success or not of pcr amplification, primer sequence designed by the present invention is guarded, suitable length, can avoid producing non-specific amplification, site distance of distance on gene according to amplification carries out design of primers simultaneously, avoid primer too much to interfere, affect amplification efficiency；This design of primers institute being far from routine is getable。
Described probe sequence is as follows:
IVS7-2P:5’-TATTTCGGACGATAATTGCTAC-3’(SEQIDNO:9)
2168P:5’-CACATTCTTTTTGACGGTCCGTG-3’(SEQIDNO:10)
1174P:5’-CCTTTGGGATCAGCTACATCTTC-3’(SEQIDNO:11)
1229P:5’-CTGCTCTTTCCCGCATGGCCGTC-3’(SEQIDNO:12)
1226P:5’-CACCACTGCTCTTTCCCACACGG-3’(SEQIDNO:13)
1975P:5’-CCAATCCATAGCCTTCTGCTTGAC-3’(SEQIDNO:14)
2027P:5’-TTGTTGGAGTGAGATCACAGCG-3’(SEQIDNO:15)
Described probe has been also respectively connected with fluorescence radiation group and fluorescent quenching group, wherein, for detecting fluorescence radiation group respectively VIC, ROX, Cy5 of the probe in IVS7-2,2168,1174 site；Fluorescence radiation group for detecting the probe in 1229,1226,1975,2027 sites is TAMRA；The fluorescent quenching group of each probe is BHQ-1。
This Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, also including 1 pair of β-Actin internal reference primer for monitoring the effectiveness expanding template and 1 internal reference probe, its sequence is respectively as shown in SEQ ID NO:16, SEQIDNO:17 and SEQIDNO:18。
Concrete, the sequence of β-Actin internal reference primer is as follows:
β-ActinF:5'-CACCCAGCACAATGAAGA-3'(SEQIDNO:16)
β-ActinR:5'-AAGGTGGACAGCGAGGC-3'(SEQIDNO:17)
The sequence of internal reference probe is:
β-ActinP:5'-CTCCTGAGCGCAAGTACTCCGTG-3'(SEQIDNO:18)
The fluorescence radiation group of described internal reference probe is FAM, and fluorescent quenching group is BHQ-1。
This Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, also includes negative Quality Control, positive quality control, and negative Quality Control is be connected into the reference gene of carrier T, normal PDS Gene Partial sequence, and its sequence is such as shown in SEQ ID NO:19；Positive quality control be connected into the reference gene of carrier T, containing IVS7-2,2168,1174,1229 mutational site partial sequence, its sequence is such as shown in SEQ ID NO:20。Setting up of Quality Control can ensure that testing result is in control, it is to avoid false negative, false-positive appearance。
Concrete, a kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, it is made up of DNA extraction liquid, PCR reactant liquor, negative Quality Control and positive quality control, wherein, consisting of of every 50 μ lPCR reactant liquors: 10 × PCR buffer 5.0 μ l, MgCl22.0～5.0mM, dNTPs0.5～1.5mM, primer 0.2～2.0 μM, probe 0.1～1.0 μM, Taq enzyme 2.0～4.0U, UNG enzyme 0.5～1.0U, add water to 50 μ l。Described primer is primer 1, primer 2, primer 3, primer 4 and internal reference primer；Described probe is 7 probes detected whether containing sudden change and 1 internal reference probe。The U-DNA before effectively removing pcr amplification that adds of UNG enzyme pollutes。
Adopting this Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit to carry out the method detected, step is:
Joining in PCR reaction tube by the DNA extraction liquid of detected sample, PCR reactant liquor, negative Quality Control and positive quality control, mixing is placed on the enterprising performing PCR amplified reaction of quantitative real time PCR Instrument, and PCR reaction condition is as follows: 50 DEG C of 2min；95 DEG C of 3min；95 DEG C of 15s, 60 DEG C of 45s, 40 circulations, each circulate in 60 DEG C gather fluorescence；RST according to amplification curve qualitatively judges whether there is sudden change。
First negative Quality Control, positive quality control testing result should be consistent with expection, namely negative Quality Control only has the amplification of FAM curve, expands without other fluorescence curve, and positive quality control FAM, VIC, ROX, Cy5, TAMRA curve all has amplification。Sample aperture just can be analyzed after being consistent with expection by only negative Quality Control, positive quality control amplification curve result。
If sample well collects FAM signal, it was shown that reference gene amplification is normal, and sample DNA is effective；
If sample well does not collect FAM signal, it was shown that reference gene expands unsuccessfully, sample DNA is invalid or instrument existing problems, and this testing result is invalid；
If sample well has FAM, VIC signal, without ROX, Cy5, TAMRA signal, it was shown that sample is IVS7-2 sudden change；
If sample well has FAM, ROX signal, without VIC, Cy5, TAMRA signal, it was shown that sample is 2168 sudden changes；
If sample well has FAM, Cy5 signal, without VIC, ROX, TAMRA signal, it was shown that sample is 1174 sudden changes；
If sample well has FAM, TAMRA signal, without VIC, ROX, Cy5 signal, it was shown that sample is a certain kind or several in 1229,1226,1975,2027 sudden changes。
Beneficial effects of the present invention:
(1) detection kit of the present invention introduces house-keeping gene actin β-Actin to compare as internal reference, design 1 is to special primer and 1 specific probe, expanding fragment length is 94bp, expand with site to be measured primer in same PCR pipe simultaneously, to detect the effectiveness of the effectiveness of sample DNA, monitoring PCR system, in addition, also can detect sample well or whether instrument has problems, it is prevented that false negative occurs。
(2) owing to 1174,1229,1,226 3 sites location comparison on PDS gene is concentrated, the present invention takes above three site is shared pair of primers, effectively prevent the interference that primer too much causes。
(3) owing to 1975,2,027 two sites location comparison on PDS gene is concentrated, the present invention takes above-mentioned two site is shared pair of primers, effectively prevent the interference that primer too much causes。
(4) present invention is respectively directed to reference gene, totally four probes in IVS7-2,2168,1174 site carry out different fluorescent labeling, reference gene-FAM, IVS7-2-VIC, 2168-ROX, 1174-Cy5, first whether effective with or without judgment sample DNA according to the specific fluorescence curve of reference gene, and then according to VIC, ROX, Cy5 specific fluorescence curve with or without whether there is sudden change in specific, concrete IVS7-2,2168,1174 site, thus the high pathogenicity rate site relevant to Delayed onset deafness is effectively confirmed；The site 12 29,1226,1975,2027 that mutation rate is low compared with IVS7-2,2168,1174 is carried out fluorescence TAMRA labelling of the same race, if reacting hole has TAMRA signal, then the mutational site of sample is a certain kind in 1229,1226,1975,2027 or several；The present invention applies different fluorophor label probes, thus not only can clearly distinguish high mutation rate site but also can detect uncommon mutational site in same PCR reaction tube, improves detection coverage rate。
(5) present invention achieves application multichannel fluorescence PCR method first and detects seven mutational sites that Delayed onset is deaf in same reaction tube simultaneously, the method is easy and simple to handle quickly, accurate, high flux, cost are low, cross-contamination is avoided in stopped pipe operation, make that deaf examination purposiveness is strong, examination scope is wide, effectively prevention Delayed onset deafness occurs, it is easy to promote and application。
Accompanying drawing explanation
Fig. 1 is the pcr amplification curve of negative Quality Control；
Fig. 2 is the pcr amplification curve of positive quality control；
Fig. 3 is normal person's sample pcr amplification curve；
Fig. 4 is IVS7-2 saltant type sample pcr amplification curve；
Fig. 5 is IVS7-2,2168 saltant type sample pcr amplification curves。
Detailed description of the invention
The present invention is further illustrated in conjunction with the embodiments, it should explanation, and its content, merely to explain the present invention, is not defined by the description below。
Embodiment 1: a kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit composition and detection method thereof
1. the composition of test kit:
DNA extraction liquid 1 manages (5.0ml), PCR reactant liquor 1 manages (1.1ml), negative (50 μ l) is managed in Quality Control 1, positive quality control 1 manages (50 μ l)；
Consisting of of every 50 μ lPCR reactant liquors: 10 × PCR buffer 5.0 μ l, MgCl23.0mM, dNTPs1.0mM, primer 1.0 μMs, probe 0.5 μM, Taq enzyme 3.0U, UNG enzyme 1.0U, add water to 50 μ l。
Wherein, primer is primer 1, primer 2, primer 3, primer 4 and internal reference primer, and its sequence is respectively as follows:
Primer 1F:5'-AACCAATGGAGTTTTTAACAT-3'(SEQIDNO:1)
Primer 1R:5'-TAAGAGGAACACCACACTCAC-3'(SEQIDNO:2)
Primer 2 F:5'-CTGGAGCAATGCGGGTTC-3'(SEQIDNO:3)
Primer 2 R:5'-GGAACCTTGACCCTCTTG-3'(SEQIDNO:4)
Primer 3F:5'-AAATACTCAGCGAAGGTCTTGC-3'(SEQIDNO:5)
Primer 3R:5'-CGAGCCTTCCTCTGTTGC-3'(SEQIDNO:6)
Primer 4F:5'-TCCCAACCAAGGAAATA-3'(SEQIDNO:7)
Primer 4R:5'-GCAATACTGGACAACCC-3'(SEQIDNO:8)
β-ActinF:5'-CACCCAGCACAATGAAGA-3'(SEQIDNO:16)
β-ActinR:5'-AAGGTGGACAGCGAGGC-3'(SEQIDNO:17)
Probe is 7 probes detected whether containing sudden change and 1 internal reference probe, and its sequence is respectively as follows:
IVS7-2P:5’-TATTTCGGACGATAATTGCTAC-3’(SEQIDNO:9)
2168P:5’-CACATTCTTTTTGACGGTCCGTG-3’(SEQIDNO:10)
1174P:5’-CCTTTGGGATCAGCTACATCTTC-3’(SEQIDNO:11)
1229P:5’-CTGCTCTTTCCCGCATGGCCGTC-3’(SEQIDNO:12)
1226P:5’-CACCACTGCTCTTTCCCACACGG-3’(SEQIDNO:13)
1975P:5’-CCAATCCATAGCCTTCTGCTTGAC-3’(SEQIDNO:14)
2027P:5’-TTGTTGGAGTGAGATCACAGCG-3’(SEQIDNO:15)
β-ActinP:5'-CTCCTGAGCGCAAGTACTCCGTG-3'(SEQIDNO:18)
Above-mentioned probe carries out fluorescent labeling, and above-mentioned probe is connected to fluorescence radiation group and fluorescent quenching group, wherein, for detecting fluorescence radiation group respectively VIC, ROX, Cy5 of the probe in IVS7-2,2168,1174 site；Fluorescence radiation group for detecting the probe in 1229,1226,1975,2027 sites is TAMRA, and the fluorescence radiation group of internal reference probe is FAM；The fluorescent quenching group of each probe is BHQ-1。
Negative Quality Control is be connected into the reference gene of carrier T, normal PDS Gene Partial sequence, and its sequence is such as shown in SEQ ID NO:19, specific as follows:
cacccagcacaatgaagatcaagatcattgctcctcctgagcgcaagtactccgtgtggatcggcggctccatcctggcctcgctgtccaccttaaccaatggagtttttaacatcttttgttttatttcggacgataattgctactgccatttcatatggagccaacctggaaaaaaattacaatgctggcattgttaaatccatcccaagggggtgagtgtggtgttcctcttaaaatactcagcgaaggtcttgcaaagattcaatttgtaggatcgttgtcatccagtctcttccttaggaattcattgcctttgggatcagctacatcttctcaggattcttctcttgttttgtggccaccactgctctttcccgcatggccgtccaggagagcactggaggaaagacacaggtaggaacaacagccttatgatatccatctcagagaacaagtcgaggaatggcaacagaggaaggctcgctggagcaatgcgggttctttgacgacaacattagaaaggacacattctttttgacggtccgtgatgct atactctatctacagaaccaagtgaaatctcaagagggtcaaggttcctcccaaccaaggaaatagagattcaagtggattggaactctgagcttccagtcaaagtgaacgttcccaaagtgccaatccatagccttgtgcttgactgtggagctatatctttcctggacgttgttggagtgagatcactgcgggtggtaaggttctggttttctgaattatacatttggagctttggcaatagtaaaatgatgtgggttgtccagtattgc
Positive quality control be connected into the reference gene of carrier T, containing IVS7-2,2168,1174,1229 mutational site partial sequence, its sequence is such as shown in SEQ ID NO:20, specific as follows:
cacccagcacaatgaagatcaagatcattgctcctcctgagcgcaagtactccgtgtggatcggcggctccatcctggcctcgctgtccaccttaaccaatggagtttttaacatcttttgttttatttcagacgataattgctactgccatttcatatggagccaacctggaaaaaaattacaatgctggcattgttaaatccatcccaagggggtgagtgtggtgttcctcttaaaatactcagcgaaggtcttgcaaagattcaatttgtaggatcgttgtcatccagtctcttccttaggaattcattgcctttgggatcagcaacatcttctcaggattcttctcttgttttgtggccaccactgctctttcccgcacggccgtccaggagagcactggaggaaagacacaggtaggaacaacagccttatgatatccatctcagagaacaagtcgaggaatggcaacagaggaaggctcgctggagcaatgcgggttctttgacgacaacattagaaaggacacattctttttgacggtccatgatgctatactctatctacagaaccaagtgaaatctcaagagggtcaaggttcc
2. detection method:
1) extracting genome DNA:
Whole blood: take 200 μ lEDTA anticoagulations to aseptic EP pipe new for 1.5ml, adds 600 μ l distilled waters, and vibration mixing, room temperature places 10min；Period mixes 2-3 time, and 12000rpm is centrifuged 5min, abandons supernatant；Add 200 μ lDNA extracting solution (before using, fully mixing, makes granule suspend), 100 DEG C of water-bath 8min；12000rpm is centrifuged 5min, takes supernatant for detecting。
Blood stasis speckle: cut-off footpath is about 3mm blood sheet in the aseptic EP pipe that 1.5ml is new, adds 500 μ l distilled waters, vortex oscillation, and room temperature places 5min；12000rpm is centrifuged 1min, abandons supernatant；Add 200 μ lDNA extracting solution (before using, fully mixing, makes granule suspend), 100 DEG C of water-bath 8min, take out vibration 15-30s；12000rpm is centrifuged 5min, takes supernatant for detecting。
2) application of sample, pcr amplification:
Subpackage 45 μ lPCR reactant liquor in PCR reaction tube, subpackage pipe number=n+2 (note: n is number of awaiting test sample, and 2 is the pipe number for negative Quality Control, positive quality control)；Being separately added into 5 μ l DNA profilings to be measured, feminine gender/positive quality control, mixing is placed on the enterprising performing PCR amplified reaction of quantitative real time PCR Instrument；Response procedures is 50 DEG C of 2min；95 DEG C of 3min；95 DEG C of 15s, 60 DEG C of 45s, 40 circulations, each circulate in 60 DEG C gather fluorescence。
3) interpretation of result:
RST according to amplification curve qualitatively judges whether there is sudden change。First negative Quality Control, positive quality control testing result should be consistent with expection, namely negative Quality Control only has the amplification of FAM curve, expands without other fluorescence curve, as it is shown in figure 1, positive quality control FAM, VIC, ROX, Cy5, TAMRA curve all has amplification, as shown in Figure 2。Sample aperture just can be analyzed after being consistent with expection by only negative Quality Control, positive quality control amplification curve result。
If sample well collects FAM signal, as shown in Figure 3, it was shown that reference gene amplification is normal, and sample DNA is effective；
If sample well does not collect FAM signal, it was shown that reference gene expands unsuccessfully, sample DNA is invalid or instrument existing problems, and this testing result is invalid；
If sample well has FAM, VIC signal, without ROX, Cy5, TAMRA signal, as shown in Figure 4, it was shown that sample is IVS7-2 sudden change；
If sample well has FAM, ROX signal, without VIC, Cy5, TAMRA signal, it was shown that sample is 2168 sudden changes；
If sample well has FAM, VIC and ROX signal, without Cy5, TAMRA signal, as shown in Figure 5, it was shown that sample is IVS7-2 and 2168 sudden changes；
If sample well has FAM, Cy5 signal, without VIC, ROX, TAMRA signal, it was shown that sample is 1174 sudden changes；
If sample well has FAM, TAMRA signal, without VIC, ROX, Cy5 signal, it was shown that sample is a certain kind or several in 1229,1226,1975,2027 sudden changes。
It has been experienced that, the Detection accuracy of the test kit of the present invention is 100%, and false positive rate is 0, and false negative rate is 0, and result is accurately and reliably。The about 1 hour detection time of test kit of the present invention, compared with existing detection kit, simple and quick；And, the required instrument price of this test kit detection is relatively low and relatively general, therefore testing cost is relatively low。
Claims (3)
1. a Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, it is characterized in that, IVS7-2A with PDS gene > G, 2168A > G, 1174A > T, 1229C > T, 1226G > A, 1975G > C, 2027T > A sports detection object, design specific primer and probe, described specific primer includes for expanding IVS7-2A > primer 1 of G, its sequence is such as shown in SEQ ID NO:1 and SEQIDNO:2；For expanding 2168A > primer 2 of G, its sequence is such as shown in SEQ ID NO:3 and SEQIDNO:4；For expanding 1174A > T, 1229C > T, 1226G > primer 3 of A, its sequence is such as shown in SEQ ID NO:5 and SEQIDNO:6；For expanding 1975G > C, 2027T > primer 4 of A, its sequence is such as shown in SEQ ID NO:7 and SEQIDNO:8；Described probe is whether the IVS7-2 of specific detection PDS gene, 2168,1174,1229,1226,1975,2027 site contains sudden change, and its probe sequence is respectively as shown in SEQ ID NO:9 to SEQIDNO:15；
Also including 1 pair of β-Actin internal reference primer for monitoring the effectiveness expanding template and 1 internal reference probe, its sequence is respectively as shown in SEQ ID NO:16, SEQIDNO:17 and SEQIDNO:18；
Described probe has been also respectively connected with fluorescence radiation group and fluorescent quenching group, wherein, for detecting fluorescence radiation group respectively VIC, ROX, Cy5 of the probe in IVS7-2,2168,1174 site；Fluorescence radiation group for detecting the probe in 1229,1226,1975,2027 sites is TAMRA；The fluorescent quenching group of each probe is BHQ-1；
Described internal reference probe is fluorescently-labeled probe, and its fluorescence radiation group is FAM, and fluorescent quenching group is BHQ-1；
Also include negative Quality Control, positive quality control；
Described negative Quality Control is be connected into the reference gene of carrier T, normal PDS Gene Partial sequence, and its sequence is such as shown in SEQ ID NO:19；Positive quality control be connected into the reference gene of carrier T, containing IVS7-2,2168,1174,1229 mutational site partial sequence, its sequence is such as shown in SEQ ID NO:20。
2. a Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit, it is characterised in that it specifically comprises: DNA extraction liquid, PCR reactant liquor, negative Quality Control and positive quality control；Wherein, the consisting of of every 50 μ lPCR reactant liquors: 10 × PCR buffer 5.0 μ l, MgCl22.0～5.0mM, dNTPs0.5～1.5mM, primer 0.2～2.0 μM, probe 0.1～1.0 μM, Taq enzyme 2.0～4.0U, UNG enzyme 0.5～1.0U, add water to 50 μ l；
Described primer is primer 1, primer 2, primer 3, primer 4 and internal reference primer, and its sequence is respectively as shown in SEQ ID NO:1 to SEQIDNO:8 and SEQIDNO:16, SEQIDNO:17；
Described probe is 7 probes detected whether containing sudden change and 1 internal reference probe, and its sequence is respectively as shown in SEQ ID NO:9 to SEQIDNO:15 and SEQIDNO:18。
3. a kind of Delayed onset deaf gene multichannel fluorescence PCR detection reagent kit as claimed in claim 2, it is characterised in that described negative Quality Control is be connected into the reference gene of carrier T, normal PDS Gene Partial sequence, and its sequence is such as shown in SEQ ID NO:19；Positive quality control be connected into the reference gene of carrier T, containing IVS7-2,2168,1174,1229 mutational site partial sequence, its sequence is such as shown in SEQ ID NO:20。
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| CN102108410A (en) * | 2010-12-22 | 2011-06-29 | 协和干细胞基因工程有限公司 | Gene combinations, primer, probe and applications thereof for determining PDS gene mutation of large vestibular aqueduct syndrome deafness |
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| CN1850992A (en) * | 2006-02-28 | 2006-10-25 | 金政策 | Method for preparing primer for in vitro determining aqueductus vestibuli syndrome deaf PDS gene mutation and its use |
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