CN104120166A - Method and kit for detecting mutation of PDS gene 2168A>G by using fluorescence PCR technology - Google Patents
Method and kit for detecting mutation of PDS gene 2168A>G by using fluorescence PCR technology Download PDFInfo
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Abstract
The invention provides a method and a kit for detecting mutation of PDS gene 2168A>G by using fluorescence PCR technology. The method comprises: aiming at a PDS 2168A>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 2168A>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 2168A > 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.2168A > G sudden change is one of modal SLC26A4 gene mutation site in EVAS patient, Chinese population is carried out to the general examination of 2168A > 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 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 2168A > G sudden change, for PDS gene 2168A > 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 2168 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 2168A > G sudden change, can be used for the detection of clinical sample.
Concrete technological line of the present invention is:
1) the 2168A > G site of PDS gene extron 19 regions (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 2168A > G site, wherein wild-type probe (SEQ ID NO:3) is for PDS gene 2168A > G wild-type site, and saltant type probe (SEQ ID NO:4) is for PDS gene 2168A > G 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 2168A > G 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 2168 site upstream design forward amplimers, 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 2168 sites 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 2168 sites.
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 2168A > 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 2168A > G site, design covers saltant type probe and the wild-type probe in 2168A > 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 '-CTTTGACGACAACATTAGAAAGG-3 '
Reverse primer (SEQ ID NO:2): 5 '-TTGACCCTCTTGAGATTTCACT-3 '
Wild-type fluorescent probe (SEQ ID NO:3): 5 ' FAM-TTTGACGGTCCATGATGCTATA-BHQ13 '
Saltant type fluorescent probe (SEQ ID NO:4): 5 ' FAM-TTTGACGGTCCGTGATGCTATA-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 1XPCR Buffer, 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 ' CTTTGACGACAACATTAGAAAGG-3 '
Reverse primer (SEQ ID NO:2): 5 '-TTGACCCTCTTGAGATTTCACT-3 '
Wild-type fluorescent probe (SEQ ID NO:3): 5 ' FAM-TTTGACGGTCCATGATGCTATA-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 2168A > 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 2168A > 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 wild homozygous tissue in PDS gene 2168A > G 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 vectors of PDS gene 2168 site mutations 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 2168A > 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 2168A > 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 2168A > 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 2168A/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 2168A/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 2168A/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 2168A > G sudden change, it is characterized in that with PDS gene the 19th exon site (2168A > 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 2168A > 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 2168A > 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 2168A > 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 2168A > 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 the 19th exon site (2168A > G) region 60-150bp fragment.
4. according to method and test kit thereof described in claim 1,2, it is characterized in that, PDS gene 2168A > 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 2168 regions, wherein wild-type probe (SEQ ID NO:3) is for PDS gene 2168A > G wild-type site, and saltant type probe (SEQ ID NO:4) is for PDS gene 2168A > 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 2168A > 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 2168A > G wild-type.
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