CN102719538A - Gene chip for non-invasive prenatal diagnosis of high-risk hereditary hearing loss and preparation method - Google Patents
Gene chip for non-invasive prenatal diagnosis of high-risk hereditary hearing loss and preparation method Download PDFInfo
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Abstract
The invention relates to a gene chip for non-invasive prenatal diagnosis of high-risk hereditary hearing loss and a preparation method in the technical field of gene diagnosis. The gene chip comprises a substrate and probes fixed on the substrate, wherein the probes are nucleotide sequences shown as SEQ ID NO.1-24. Usage of the gene chip includes the steps of sample preparation, multiplex PCR (polymerase chain reaction) amplification, ligase detection reaction, chip hybridization, chip scanning and result obtaining. Beside, the invention further relates to a reagent kit comprising the gene chip. The gene chip for diagnosis is simple in operation steps, only requires the multiplex PCR reaction, the ligase detection reaction and hybrid scanning once, is high in detection specificity and good in stability so as to be capable of correctly distinguishing homozygotes from heterozygotes of each locus and high in repeatability of repeated experiments, is short in detection time and low in cost as the substrate can be universally used for other chips by means of universal chip technology.
Description
Technical field
The invention belongs to the gene diagnosis technical field, be specifically related to the gene chip and the method for use of the high-risk hereditary hearing impairment of a kind of non-invasive prenatal diagnosis.
Background technology
Deafness is one of modal disabling disease and clinical inherited disease, and newborn infant's sickness rate of having reported reaches 1～3 ‰, discovers, about 60% newborn infant is deaf relevant with inherited genetic factors, i.e. hereditary hearing impairment.Wherein about 70% with the heredity of autosomal recessive mode.Hereditary hearing impairment is caused by term single gene sudden change or heterogeneic complex mutation.So far individual surplus the deaf-related gene of finding has reached 120, have higher gene and site genetic heterogeneity.Recent study finds that genes such as GJB2, SLC26A4 are modal deaf-related genes; 235delC, 299delAT, 176del16 that GJB2 is last; And the IVS7-2A on the SLC26A4>G, 2168A>G etc. is hot mutant site, pathologic carrying rate and diallele mutation rate are all high.Carry out examination to above-mentioned focus sudden change, can be the clear and definite cause of disease of deaf patient more than 40%.
Hereditary hearing impairment multilist clinically is the sensorineural deafness of severe at present, lacks effective clinical treatment way, so early diagnosis, early prevention are main hereditary hearing impairment prevention and treatment methods.It is a kind of technique means of effective solution hereditary hearing impairment morbidity that antenatal diagnosis combines gene test.The principle of antenatal diagnosis technology is for making diagnosis as early as possible, and adopts safe intervention property methods for prenatal diagnosis as far as possible, mainly comprises invasive and Noninvasive dual mode.
(chorionic villus sampl ing, CVS), amniocentesis is representative, diagnoses on gene level because of it, accuracy rate is up to 99% with the fine hair biopsy in the invasive antenatal diagnosis.Traditional antenatal diagnosis hereditary hearing impairment uses the invasive prenatal gene diagnosis exactly, and common deaf genes such as fetus row GJB2, SLA26A4 are detected.But because processes such as cell cultures, sense cycle reached for 1～2 week, and had miscarriage, fetal anomaly equivalent risk, and this technology only limits to the diagnosis of high risk gravida at present, is unfavorable for promoting on a large scale.
Non-invasive prenatal diagnosis has serology detection, iconography detection, genetics detection etc.There are higher false positive and false negative possibility in serology and iconography means owing to be the inspection to presentation; And the detection of emerging genetics mainly is that the fetus genetic material in the maternal blood is carried out gene diagnosis; Accuracy is high; Compare with the invasive methods for prenatal diagnosis, only need the pregnancy period to extract an amount of maternal blood and can realize detecting, avoided miscarriage, fetal anomaly equivalent risk; The round pcr that detection relates to is swift in response, sense cycle only 1～2 day; Satisfy antenatal diagnosis and diagnose principle in early days, safely, fast.
The method that at present adopts the Noninvasive means to carry out Genetic Detection mainly contains: extract that the maternal peripheral blood fetal nucleated red blood detects or enrichment maternal peripheral blood free fetal dna (cell free-fetal DNA cf-fDNA) detects.The former can obtain the fetus complete genome DNA, but because fetal cell quantity is few, separation, discriminating and beneficiation technologies are complicated, and expensive has limited clinical application, at present still in the experimental study stage; Latter's foetal DNA content is more relatively, gestation early stage be respectively late period 775 times and 970 times of fetal cell DNA in female blood, maternal peripheral blood can satisfy the diagnosis needs on a small quantity.In addition, because the pregnant cf-fDNA (the earliest can behind the zygote implantation the 18th day record) that can in peripheral blood, detect in early days, and divide the puerperium to remove rapidly, be beneficial to pregnant early stage genetics antenatal diagnosis.At present, cf-fDNA has been used for clinical detection such as the analysis of RhD blood group, sex identification, and a large amount of explorations are also being arranged aspect chromosomal disorder, the monogenic disease diagnosis, and effect is remarkable.
Therefore, the Non-invasive Prenatal Diagnosis technology based on parent blood plasma cf-fDNA can realize in early days, diagnose safely, fast inherited disease on gene level.For hereditary hearing impairment, this technology can remedy the limitation of UNHS, the delayed deafness that promptly can't find through examination; Can solve the limitation of traditional deaf invasive antenatal diagnosis, promptly have miscarriage, fetal anomaly risk, sense cycle is long, deficiency such as is difficult to carry out on a large scale; " not sick diseases prevention " accomplished in the recessive mutation of carrying in can the examination crowd; For carrying out of fetal in utero treatment in the future lays the foundation.
Because maternal blood DNA is the mixture of fetus and parent dissociative DNA, and foetal DNA content is merely 3.4～6.2%.Parent equipotential dna fragmentation can disturb the detection of foetal DNA.Therefore seek the problem that detection means highly sensitive, that specificity is good becomes needs urgent solution.Traditional detection means such as restriction fragment length polymorphism analysis (Restriction Fragment Length Polymorphism; RFLP), single-strand conformation polymorphism analysis (Single Stranded Conformational Polymorphism; SSCP), covert efficient liquid phase chromatographic analysis (Denaturing High Performance Liquid Chromatography; DHPLC), mass spectroscopy (Mass Spectrometry; MS) complicated operation, expensive, poor accuracy such as, and can not high-throughput, carry out Polymorphism Analysis on a large scale, be difficult to satisfy requirements for clinical application.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, the gene chip and the method for use of the high-risk hereditary hearing impairment of a kind of non-invasive prenatal diagnosis is provided.Diagnosing chip typing method operation steps of the present invention is simple, and detection specificity is high, good stability, and the time is short, and cost is low, can be used for the detection of low abundance hereditary hearing impairment common mutations, has great potential in the non-invasive prenatal diagnosis field.
Technical scheme of the present invention is to realize through following technical scheme,
First aspect the present invention relates to the gene chip of the high-risk hereditary hearing impairment of a kind of non-invasive prenatal diagnosis, comprises the sheet base and be fixed on the probe on the sheet base that said probe is the nucleotide sequence shown in SEQ ID NO.1～24.
Preferably, said base is slide glass, silicon chip or film.
Preferably, the material of said base is a macromolecular material.
Second aspect the present invention relates to aforementioned gene chip preparation method, comprises the steps: sheet base acid-base pretreatment, and amination is handled, the isothiocyanic acid processing, and chip Zip probe design, Zip probe point sample, the ammoniacal liquor sealing promptly gets.
The third aspect the present invention relates to the method for use of aforementioned gene chip, comprises the steps: to prepare sample, the multiplex PCR amplification, and the ligase enzyme detection reaction, chip hybridization, chip scanning obtains the result.
Preferably, in the amplification of said multiplex PCR used primer to as shown in the table:
Fourth aspect the invention still further relates to a kind of test kit that is used for the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis, and said test kit comprises aforesaid gene chip.
Gene chip based on maternal blood cf-fDNA non-invasive prenatal diagnosis hereditary hearing impairment of the present invention; Be to be template with maternal blood dissociative DNA to be measured (mixture of fetus and parent dissociative DNA); Carry out the multiplex PCR amplification with the corresponding allelic primer sets in hereditary hearing impairment common mutations site; With the multiplex PCR amplified production and the chip hybridization that obtain, confirm according to results of hybridization whether fetus carries the allelotrope in mutational site.
The present invention has following beneficial effect: diagnosing chip operation steps of the present invention is simple, only need carry out multi-PRC reaction and ligase enzyme reaction, and hybridization scans and gets final product; Detection specificity is high, good stability, and this chip can correctly be distinguished the homozygote and the heterozygote in each site, and test of many times repeatability is high; Time is short, can in a working days, accomplish to obtaining scanning result from the sample extracting; Employing general-purpose chip technology, the sheet base also can be common to other chips, effectively reduces cost, is applicable to that clinical patients detection in Gene Mutation, antenatal diagnosis and normal people's heterozygote carrier detect.
Description of drawings
Fig. 1 is the detected result to GJB2-235delC site wild-type;
Fig. 2 is the detected result to GJB2-235delC site mutation type.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Be interpreted as: these embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; Sambrook equimolecular clone for example: laboratory manual is seen the condition described in the New York Cold Spring Harbor Laboratory press version in 1989, or the condition of advising according to manufacturer.
Gene chip of the present invention adopts based on ligase enzyme detection reaction (Ligase Detect Reaction; LDR) classifying method that combines with general-purpose chip (Universal Array) technology; To 8 hot mutant site of hereditary hearing impairment among the crowd, 3 probes of each site design.Wherein two are positioned at the mutational site and the upper reaches thereof, and its 5 ' end is respectively wild-type and mutant with mutational site paired base, is called specific probe; Article one, be positioned at the downstream, mutational site, match fully with template sequence, be called the common probe, its 5 ' end carries fluorophor.Have only the specific probe of working as and common probe sequence all to match fully with template, ligase enzyme just can couple together two probes; If there is base mismatch, ligation then can not be carried out.Therefore, have only specific probe ability and the common probe identical to link together, thereby send fluorescent signal with the templet gene type.Be fixed with the Zip probe on the chip slapper base; Specific probe 3 ' end has the sequence with Zip probe sequence complementary cZip; So when reacted product of LDR and chip hybridization; The cZip sequence just can guide reaction product and Zip probe hybridization, can detect fluorescent signal at the specific position of chip, thereby analyzes the genotype of template.
Gene chip of the present invention is the carrier in mutational site of the same race not on the hereditary hearing impairment Disease-causing gene seat for father and mother both sides; Can diagnose and confirm whether fetus carries property mutational site in father source on this locus, the Disease-causing gene that can clear and definite father whether will be carried on the karyomit(e) passes to filial generation.For this autosomal recessive hereditary diseases of hereditary hearing impairment, father and mother both sides are heterozygote sudden change person, if detect property mutational site, father source, then fetus has 50% risk to suffer from this disease, and the invasive antenatal diagnosis of should further going is clear and definite; If do not detect property mutational site, father source, then fetus can get rid of the morbidity possibility, but 50% the carrier that possibly become the Disease in Infants sudden change is arranged.
Before exercising traditional methods for prenatal diagnosis, use gene chip of the present invention can confirm whether fetus is high-risk hereditary hearing impairment infant; Specifically; If chip results shows the mutation allele that does not occur the father source in the parent sample; Then fetus heredity is the normal allelotrope of father, so just can avoid the antenatal diagnosis of fetus row invasive; If chip results shows the mutation allele that occurs the father source in the parent sample; Then the genotype of fetus shows as heterozygous mutant type or homozygous mutation type; The risk of suffering from hereditary hearing impairment is higher, needs further to confirm through traditional invasive methods for prenatal diagnosis whether fetus is ill.
The advanced part of this chip is that also only the disposable extraction pregnant woman's of need whole blood can detect the genotype of mother and fetus simultaneously on a chip.Specifically; Be designed with two on the chip of the present invention and go up the appearance district; The pregnant woman's whole blood part that extracts is used for the total DNA of extracting maternal blood; The DNA that a part is used for the extracting plasma free distinguishes the gene type that can be maternal gene group DNA for one last kind, and the gene type of cf-fDNA can be done by another district.Such chip can detect the genotype of mother and each pathogenic sites of fetus hereditary hearing impairment simultaneously, can judge the ill risk of fetus according to the genotype situation of different pathogenic sites, can also save unnecessary flow process and expense.
The preparation of the gene chip of embodiment 1, the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis
Present embodiment relates to the gene chip of the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis, and this gene chip preparation method comprises the steps:
Slide is cleaned with distilled water, and soaked overnight in alkali lye is taken out the back and is cleaned 3 times with distilled water, in volume(tric)fraction is 1% HCl, soaks 30 minutes again, clean be placed in the slide cylinder for use.Slide after the cleaning through 4-ammonia butyl triethoxyl silane solution amination processing, is carried out the isothiocyano processing through the phenylthioisocyanate ester solution again, after drying up with nitrogen, be put in 4 ℃ and keep in Dark Place;
Chip Zip probe is that the length that makes up at random is the oligonucleotide fragment of 24bp; Utilize BLAST function and goal gene group sequence on the NCBI website to carry out homology relatively these oligonucleotide sequences, select minimum one group of homology degree wherein as the Zip probe sequence.The locus specificity probe length does not wait, and needs to guarantee that its Tm value is consistent basically, to guarantee the homogeneity of subsequent reactions;
Synthetic good Zip probe is dissolved in the sampling liquid, probe solution is transferred in the 96 corresponding orifice plates, use the GMS417 point sample instrument; The operation corresponding program with probe points to the good sheet base of activation; Place 37 ℃, spend the night in the climatic chamber of 90% humidity, make probe and slide full cross-linked.The reactive group of the sheet base capping in ammoniacal liquor that fixes can be stored in 4 ℃ to get step hybrid experiment usefulness ready.Those skilled in the art know, and present embodiment is used to diagnose the gene chip of high-risk hereditary hearing impairment to cooperate conventional detection reagent, promptly can obtain can being used to diagnose the test kit of high-risk hereditary hearing impairment, the use that routinizes.
Embodiment 2, utilize the high-risk hereditary hearing impairment of gene chip non-invasive prenatal diagnosis
2.1 sample is prepared
Prepare plasma sample, with QIAamp Circulating Nucleic Acid Kit (Qiagen, Basel, Switzerland) free DNA in the extracting blood plasma.
2.2 the selection of sudden change detection site
8 mutational sites need detecting and probe core sequence such as table 1,
Table 1
" del " expression deletion mutantion in the mutant form is represented the 35th bases G disappearance in the nucleotide sequence shown in the SEQ ID NO.25 like 35delG; ">" the expression replacement mutation, like 707T>C representes that the 707th base sports C by T in the nucleotide sequence shown in the SEQ ID NO.26.By that analogy, more than in 8 kinds of sudden changes the position of preceding 5 kinds of representatives be in the nucleotide sequence shown in the SEQ NO ID.25:
Whether the 35th base undergos mutation, the 35th bases G disappearance;
Whether the 167th base undergos mutation, the 167th base T disappearance;
Whether the 176th to 191 base undergos mutation, the 176th to 191 these 16 base deletions;
Whether the 235th base undergos mutation, the 235th base C disappearance;
Whether the 299th to 300 base undergos mutation, the 299th to 300 these two base AT disappearances;
More than in 8 kinds of sudden changes the position of the 6th kind and the 7th kind representative be in the nucleotide sequence shown in the SEQ ID NO.26:
Whether the 707th base undergos mutation, by T → C; Whether the 2168th base undergos mutation, by A → G;
More than in 8 kinds of sudden changes the position of the 8th kind of representative be whether to undergo mutation in the 99th base of the nucleotide sequence shown in the SEQ ID NO.27, by A → G;
The detected result of said probe is following:
Whether the 35th base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.1-3 SEQ ID NO.25 undergos mutation the 35th bases G disappearance;
Whether the 167th base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.4-6 SEQ ID NO.25 undergos mutation the 167th base T disappearance;
Whether the 176th to 191 base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.7-9 SEQ ID NO.25 undergos mutation the 176th to 191 these 16 base deletions;
Whether the 235th base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.10-12 SEQ ID NO.25 undergos mutation the 235th base C disappearance;
Whether the 299th to 300 base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.13-15 SEQ ID NO.25 undergos mutation, the 299th to 300 these two base AT disappearances;
Whether the 707th base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.16-18 SEQ ID NO.26 undergos mutation, by T → C;
Whether the 2168th base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.19-21 SEQ ID NO.26 undergos mutation, by A → G;
Whether the 99th base of nucleotide sequence shown in the corresponding detection of the Nucleotide shown in the SEQ ID NO.22-24 SEQ ID NO.27 undergos mutation, by A → G.
2.3 multiplex PCR amplification
Dissociative DNA with each sample is a template respectively, carries out multiplex PCR.
For preventing overlapping between interaction and the amplified fragments between some primer, multiplex PCR carries out in two pipes.
Wherein, No. 1 pipe sixfold PCR reacted constituent (15 μ l system): the 20ng genomic dna, 1 * QIAGEN Multiplex PCR Master Mix, 0.2 each primer of μ M, RNase-free water supplies;
No. 2 pipe fragment reacted constituents (15 μ l system): the 20ng genomic dna, 1 * Lay maple PCR Master Mix, 0.4 μ M primer, RNase-free water supplies.
The reaction conditions of No. 1 pipe sixfold PCR is: 95 ℃ of initial 15min, and 94 ℃ of sex change 30s, 60 ℃ of annealing 90s, 72 ℃ are extended 1min, circulate 35 times, and 72 ℃ are extended 10min;
Fragment amplification reaction conditions of No. 2 pipes is: 94 ℃ of initial 5min, and 94 ℃ of sex change 30s, 64 ℃ of annealing 30s, each circulation descends 0.5 ℃; 72 ℃ are extended 1min, circulate 94 ℃ of sex change 30s, 57 ℃ of annealing 30s 14 times; 72 ℃ are extended 1min, circulate 30 times, and 72 ℃ are extended 10min.
After accomplishing, reaction in reaction mixture, adds 1 μ l Proteinase K, 70 ℃ of reactions 10min, 94 ℃ of 20min inactivated proteases K subsequently; The primer of multi-PRC reaction is as shown in table 2,
Table 2
2.4 ligation
Amplified production behind the protease K digesting carries out the LDR reaction at once.In reaction solution, add 10 * Taq ligase buffer, 2 μ l successively, probe mixed solution 1 μ l, each concentration and probe concentration is 1 μ M, Taq DNA ligase 0.25 μ l, moisturizing to TV 20 μ l; Reaction conditions is 94 ℃ of sex change 30s, and 65 ℃ of annealing 4min circulate 40 times.
2.5 hybridization
In 20 μ l LDR reaction solutions, add 15 μ l distilled waters and 35 μ l, 2 * hybridization solution, in 95 ℃ of heat denatured 5min, be put in cooled on ice rapidly behind the mixing, centrifugal for use.
To hybridize the sticking card of frame in the point sample zone, and draw 65 μ l hybridization solutions and add in the hybridization frame, and seal and place 65 ℃ of hybridization casees to hybridize 2 to 3 hours or spend the night.
Hybridization is removed the hybridization frame after finishing, and chip is put in each wash-out 1min among 0.3 * SSC/0.1%SDS and the 0.06 * SSC, centrifuge dripping respectively.
2.6 scanning and analysis
Chip after the hybridization scans with the laser co-focusing microarray scanner, and the image that obtains is analyzed with software.Finally obtain the fluorescence signal intensity value and the background value in each sample site, obtain the AMSI value (absolute median signal intensities) of this point after the influence of background correction value.According to the absolute strength ratio of same site wild-type and mutant probe points, confirm genotype.Chip scanning part as a result is as depicted in figs. 1 and 2, and Fig. 1 is the detected result to GJB2-235delC site wild-type; Fig. 2 is the detected result to GJB2-235delC site mutation type.As shown in Figure 1, point out the mutation allele that does not contain father source, this site in the maternal sample; As shown in Figure 2, point out the mutation allele that contains the father source in the maternal sample, the detection information of comprehensive all sites can judge that fetus suffers from the risk of hereditary hearing impairment.
In sum, the operation steps of present embodiment is simple, only need carry out multi-PRC reaction and ligase enzyme reaction, and hybridization scans and gets final product; Detection specificity is high, and good stability is tested the genotype that this chip can correctly be distinguished each site premenstruum, and test of many times repeatability is high; Time is short, and the sample extracting can be accomplished in a working days to obtaining scanning result; Employing general-purpose chip technology, the sheet base also can be common to other chips, effectively reduces cost.
Claims (7)
1. the gene chip of the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis comprises the sheet base and is fixed on the probe on the sheet base, it is characterized in that, said probe is the nucleotide sequence shown in SEQ ID NO.1～24.
2. the gene chip of the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis as claimed in claim 1 is characterized in that, said base is slide glass, silicon chip or film.
3. the gene chip of the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis as claimed in claim 1 is characterized in that, the material of said base is a macromolecular material.
4. the gene chip preparation method of the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis as claimed in claim 1 is characterized in that, comprises the steps: sheet base acid-base pretreatment; Amination is handled; The isothiocyanic acid processing, chip Zip probe design, Zip probe point sample; The ammoniacal liquor sealing promptly gets.
5. the method for use of the gene chip of the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis as claimed in claim 1 is characterized in that, comprises the steps: to prepare sample; The multiplex PCR amplification, ligase enzyme detection reaction, chip hybridization; Chip scanning obtains the result.
6. the method for use of the gene chip of the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis as claimed in claim 5 is characterized in that, primer used in the said multiplex PCR amplification is to as shown in the table:
7. a test kit that is used for the high-risk hereditary hearing impairment of non-invasive prenatal diagnosis is characterized in that, said test kit comprises the described gene chip of claim 1.
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