CN101684496A - Kit for detecting susceptibility genes of autism of children - Google Patents
Kit for detecting susceptibility genes of autism of children Download PDFInfo
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- CN101684496A CN101684496A CN200810200630A CN200810200630A CN101684496A CN 101684496 A CN101684496 A CN 101684496A CN 200810200630 A CN200810200630 A CN 200810200630A CN 200810200630 A CN200810200630 A CN 200810200630A CN 101684496 A CN101684496 A CN 101684496A
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Abstract
A kit used for detecting susceptibility genes of autism of children can overcome the limitations of behavior diagnosis methods. The kit comprises a DNA extraction reagent, premixed PCR reaction liquid, a standard positive control substance and a standard negative control substance. When the kit is used, the DNA samples of the children to be detected are taken as the detecting objects, after the sample DNA is extracted, the kit is used for reaction on a fluorescence quantitative PCR instrument and diagnosis is carried out in combination with the reaction results. The kit of the invention only takes DNA samples of children, such as oral samples, and analyzes and detects the samples, is convenient, objective and quick, is not subject to age and individual difference, can be used for the auxiliary diagnosis detection method of autism of infants and is quite significant in early discovery and early intervention of autism.
Description
Technical field
The present invention relates to a kind of diseases predisposing gene testing product, specifically, relate to a kind of relevant kit for detecting susceptibility genes of autism of children.
Background technology
Autism of children claims childhood autism again, be a class with serious lonely, lack emotional responses, development of speech obstacle, mechanical palikinesia and peculiar reaction is the mental disorder of feature to environment.Be referred to as autistic disorder now again.It belongs to the ubiquity dysplasia.In the origin cause of formation, development pattern and treatment means and adult autism very big difference is arranged all, it is a kind of serious infant development obstacle.Associate the unusual of three aspects with action of social interaction, language and behavior, and be feature with onset before three years old.Usually before betiding 3 years old, be more common in boy.Generally will show before 3 years old, occur since infancy, continue up to all the life, be a kind of disease of serious mood entanglement.
Autism does not have the branch of race, society, religion, has nothing to do with family income, mode of life, education degree.According to the European and American countries statistics, among per approximately 10,000 children the 2-13 example is arranged.At present, estimate 500,000 left and right sides autism infants are arranged approximately in China.This disease is more common in boy, and M-F is 2.6-5.7: 1.The Beijing Medical University mental health 1989-1992 of institute adds up in 60 examples, the man: the woman is 7.5: 1, and the man is added up in 19 examples in nineteen ninety-five-1997 a year infantile psychology outpatient service: the woman is 6.3: 1.In June, 1984 to 1986, year Nanjing infantile psychology in a May health research centre hospitalized from 170 of a lot of geographic psychosis infants in the whole nation, wherein diagnosed autism 15 examples, accounted for 8.82%, and this numeral is only represented recall rate among the children that suffered from mental disorder.
The performance of childhood autism (symptom) is as follows:
Cardinal symptom is exactly not associate and set up normal social relationships with others.The infant calmness can't be linked up, exchange in the world of oneself with language, expression, the action father and mother with others even oneself.It is retarded or introverted that the child who has can be mistaken as when beginning, and it is very normal that the child who also has seemed in the time of one or two years old, and just finding by about 3 years old has the foreign peoples to show.Autistic patients study normal people's language can be very difficult, exchanges with the people and also very difficult with extraneous communication, and they may repeat several actions (clap hands, wave).Occur changing in daily life, they can resist strongly.Autism is to the influence of behavior, except language and social difficulty, shows very excitedly or dejected in face of also having father and mother, household.
Specifically:
1. early stage performance, extremely lonely, can not smile to relatives.When breast-feeding, infant is not close to the adult with body.Stretch out one's hand when embracing, infant does not have the posture of meeting, and can not stretch out one's hand and do preparation by embracing, body can not pressed close to mother, and eyes are not seen the people who embraces him yet.
2. social difficulty, lonely especially, lack contacts with the people, lack the emotion contact, even father and mother are not reluctant to leave yet, as the stranger.But get along with the stranger, do not feel again to shrink.The normal child often expresses oneself emotion and requirement to stare the other side, and infant lack with human eye to the staring of eye, can not give expression to one's sentiment by this way and requirement.Do not like to play, do not like to play games.About infant to 5 year old, friend Chang Haiwu seldom plays with child, lacks emotional responses, usually says or makes some and do not conform to social things and come.
3. the slow or obstacle of development of speech.Infant is kept silent usually, or is echo-speech in a minute, as the mimicker's that repeats the words of others like a parrot language.To language to understand ability to express low, can't understand complicated a little any sentence, expression " goodbye " can not use gesture.Not will appreciate that and use facial expression, action, attitude and tone etc. to associate with the people.Lack imagination and social simulation, can not as the normal child go with toy " cook ", " running a train ", " putting up a house ".The infant language that has is mechanical, and pronoun is misused, and says " you want " as " I will ", or oneself is called him.Exchange difficulty with the external world, new word and its implication can not be linked, use gesture rather than language exchanges with the people.
4. ceremony and compelling sex behavior.Change and imagination owing to lack, infant usually adheres to repeating mechanical game mode, repeats some physical task, repeat identical life, to the toy queuing, play with the toe of oneself always as repeatedly, the order of wearing the clothes is identical, adheres to the form of putting of some object, can not change.In case change to some extent, they can be very dejected, others changes, and infant is just made a scene.Any variation to own room also all can make difficulties and uneasiness, adds the displacement of furniture, the variation of ornament etc.
5, brain intelligence is lower than the normal people mostly, has only people's IQ of 20% to be higher than normal people or suitable with the normal people.
6,, as a cup, a brick, express particular interest, even produce attachment, and relatives are not produced attachment to some object.
In addition, the infant that has also has disturbance of perception, and is blunt or irritated to polyesthesias such as looking, listen, touch.What have exists cognitive disorder, mental retardation, and abstract thinking ability is very poor, and the minority infant may be with epileptic seizures.It is deaf sometimes to suffer from autistic child, and sound is not had reaction.Normal child can be by the sound scaring of for example barking, and the autism child can remain indifferent or apathetic.They do not like to make friends to pain, cold and hot yet not too responsive, would rather be alone, and seldom can contact others' eyes or laugh at.
The most important thing is education and behavior therapy in the treatment, purpose is the rectification that promotes the education, particularly social behavior of infant normal behaviour, corrects abnormal behaviour, as mechanical flag etc., eliminates somnopathy, flies into a rage, Secondary Symptom such as moving etc. how.The kinsfolk of infant also will note overcoming anxiety, self-accusation, rashness, can produce good effect to the treatment of infant.
For the autism infant, have only discovery morning, morning to intervene, carry out behavior and rescue, could help them to shorten and normal social gap, allow them be socially reintegrated early.In case miss the optimal treatment phase, family is tantamount to the heavy persistent disaster of a field depth because will allow degree of depth autism children get back to " human world " from " paradise ", perhaps be very long, do not have a wait of the end forever.How could find child's autism tendency in early days?
Because society strange to autism, early stage autism children often is considered to unsociable and eccentric or " opening evening ", thereby has incured loss through delay treatment opportunity.Judgement is made in the behavior that at present mainly is the statistical study child clinically.But because the immature development of baby's behavior own, and individual difference is arranged; Add the difference of temperament, judge relatively more subjective, also more consuming time comparatively speaking.
Therefore,, can be used for the detection method of infant autism auxiliary diagnosis, for finding that it is very significant early intervening the morning of autism if provide a kind of more objective.
Summary of the invention
For can be easy and objectively autism of children is tested, the invention provides a kind of kit for detecting susceptibility genes of autism of children, can be test sample book with children's dna sample, tumor susceptibility gene to autism of children detects, thereby, provide important reference information for prevention and diagnosis autism of children.
This test kit is by the DNA extraction agent, pre-mixed PCR reaction solution, and standard positive reference substance, standard negative reference substance is formed.Dna sample with tested children when using is detected object, after the sample DNA extracting, uses test kit to react on quantitative real time PCR Instrument, and association reaction is the result diagnose.
Described pre-mixed PCR reaction solution contains SYBR green I, and the relevant OXTR-1 of autism of children and the amplimer of OXTR-2 gene mutation site; Amplimer is upstream primer and the downstream primer that comprises the mutational site, and sequence is as follows respectively:
The primer of amplification gene OXTR-1:
Upstream primer F1A 5-GAA GCC CCG CAAACC GA-3 17bp,
Upstream primer F1G 5-GAA GCC CCG CAAACC GG-3 17bp,
Downstream primer R1 5-CAG TGC ACA GCC TGAACAGC-3 20bp;
The primer of amplification gene OXTR-2:
Upstream primer F2A 5-CCA CGAACG GAT ATG CGGA-3 19bp,
Upstream primer F2G 5-CCA CGAACG GAT ATG CGG G-3 19bp,
Downstream primer R2 5-CTG GGAATG GGA CAA GCA CG-3 20bp.
Above-mentioned kit for detecting susceptibility genes of autism of children, described positive reference substance have 4, comprise following sequence respectively:
SEQ1：
TTGTGTGGGC?TGCTTTGTGG?CACAACTCCA?GGGGATGCCT?TTTACCTCAT
GGTCTATGAA?CAGGGTGTTC?CTAGGAGCTT?TGCAGTGCAC?AGCCTGAACA
GCTGAACATG?GCAGCCTCAT?CCAGTGCCCC?TTTCAGGAAA?CCATCCCTGT
TTTCTCAGTT?TGCGGGGCTT?CTTCCTCTGA?ATGCTAAGTC?AT；
SEQ2：
ATGACTTAGC?ATTCAGAGGA?AGAAGCCCCG?CAAACTGGGA?AAACAGGGAT
GGTTTCCTGA?AAGGGGCACT?GGATGAGGCT?GCCATGTTCA?GCTGTTCAGG
CTGTGCACTG?CAAAGCTCCT?AGGAACACCC?TGTTCATAGA?CCATGAGGTA
AAAGGCATCC?CCTGGAGTTG?TGCCACAAAG?CAGCCCACAC?AA；
SEQ3：
GGCTCCACTC?CTGGAGACTC?CACGAACGGA?TATGCTGAGT?CCACCGTGAA
ACAAACCGGG?AGGGCCGTGA?GGAGACCGCC?GCGTTTCTCT?TCAGACGCGG
GTAGGGCGTG?CTTGTCCCAT?TCCCAGGAAC?CCAACTCATC?TGAAACAACA
GGGCACAACC?GCCGGCCTCG?GGTTGCTCAG?CCGCCACCCC?AG；
SEQ4：
CTGGGGTGGC?GGCTGAGCAA?CCCGAGGCCG?GCGGTTGTGC?CCTGTTGTTT
CAGATGAGTT?GGGTTCCTGG?GAATGGGACA?AGCACGCCTT?ACCCGCGTCT
GAAGAGAAAC?GCGGCGGTCT?CCTCACGGCC?CTCCCGGTTT?GTTTCACGGT
GGACCCAGCA?TATCCGTTCG?TGGAGTCTCC?AGGAGAGGAG?CC。
Above-mentioned kit for detecting susceptibility genes of autism of children, the sequence that described negative control product comprise is:
SEQ5：
TTCGTTATTG?AATCTTGTTT?TGGTTCTCTT?GTGTGGAATG?CATTGTTCTT
GCTTCTCTGG?AACTTGGGAT?ATGATGACAA?AAATACGATA?TCTCTAAGGC
TAATTGCTGT?GCGGATGAAA?TATATTTGCA?CGTCAGTCTG?ATCCCATTAT
GTATATAACA?TTAGGAGTTG?GTGCATGCTC。
The dna sample that use of the present invention only need be got children carries out analyzing and testing, and is such as the oral cavity sample, convenient, objective, quick.Be not limited by age and individual difference.The detection method that can be used for infant autism auxiliary diagnosis is for finding that it is very significant early intervening the morning of autism.
Description of drawings
Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 are the amplification curve diagram on the quantitative real time PCR Instrument.The DeltaRn vs Cycle of each figure top is meant the logarithmic amplification collection of illustrative plates of Δ Rn with circulation change; Δ Rn (Delta Rn) is meant the logarithmic value at one group of given following fluorescent signal that generates of PCR condition.The X-coordinate of this figure is represented cycle number (Cycle Number), and ordinate zou is represented fluorescent value (Delta Rn).Horizontal line is represented fluorescence thresholding, and the pairing cycle number in the point of crossing of curve and fluorescence thresholding curve is the Ct value.
Fig. 1 represents the product amplification curve diagram of the homozygous sample of OXTR-1 gene GG: the Ct cycling numerical value that thresholding is represented to reach in curve 1 and the point of crossing of fluorescence thresholding curve is C1, and the Ct cycling numerical value that thresholding is represented to reach in curve 2 and the point of crossing of fluorescence thresholding curve is C2.
Fig. 2 represents the product amplification curve diagram of the homozygous sample of OXTR-1 gene A A: the Ct cycling numerical value that thresholding is represented to reach in curve 1 and the point of crossing of fluorescence thresholding curve is C3, and the Ct cycling numerical value that thresholding is represented to reach in curve 2 and the point of crossing of fluorescence thresholding curve is C4.
Fig. 3 represents the product amplification curve diagram of OXTR-1 Gene A heterozygous sample: the Ct cycling numerical value that thresholding is represented to reach in curve 2 and the point of crossing of fluorescence thresholding curve is C5, and the Ct cycling numerical value that thresholding is represented to reach in curve 1 and the point of crossing of fluorescence thresholding curve is C6.
Fig. 4 represents the product amplification curve diagram of the homozygous sample of OXTR-2 gene A A: the Ct cycling numerical value that thresholding is represented to reach in curve 5 and the point of crossing of fluorescence thresholding curve is C7, and the Ct cycling numerical value that thresholding is represented to reach in curve 6 and the point of crossing of fluorescence thresholding curve is C8.
Fig. 5 represents the product amplification curve diagram of OXTR-2 Gene A heterozygous sample: the Ct cycling numerical value that thresholding is represented to reach in curve 5 and the point of crossing of fluorescence thresholding curve is C9, and the Ct cycling numerical value that thresholding is represented to reach in curve 6 and the point of crossing of fluorescence thresholding curve is C10.
Fig. 6 represents the product amplification curve diagram of the homozygous sample of OXTR-2 gene GG: the Ct cycling numerical value that thresholding is represented to reach in curve 6 and the point of crossing of fluorescence thresholding curve is C11, and the Ct cycling numerical value that thresholding is represented to reach in curve 5 and the point of crossing of fluorescence thresholding curve is C12.
Specific embodiment:
Below in conjunction with specific embodiment, further set forth the present invention.
Be to be understood that, unaccounted normal condition and method among the following embodiment, usually according to the conventional employing method of affiliated field experimenter: as " molecular cloning experiment guide " third edition of Sa nurse Brooker and Russell chief editor, or the step and the condition of advising according to manufacturer.
Embodiment 1:
The preparation of test kit.Comprise the preparation of (1) probe; (2) DNA extraction agent; (3) premix reaction solution; (4) standard positive control; (5) standard negative control.
(1) preparation of probe;
Synthetic by common nucleotide primer synthesizer.Sequence is as follows:
The primer of amplification gene OXTR-1:
Upstream primer F1A 5-GAA GCC CCG CAA ACC GA-3 17bp,
Upstream primer F1G 5-GAA GCC CCG CAA ACC GG-3 17bp,
Downstream primer R1 5-CAG TGC ACA GCC TGA ACA GC-3 20bp;
The primer of amplification gene OXTR-2:
Upstream primer F2A 5-CCA CGA ACG GAT ATG CGG A-3 19bp,
Upstream primer F2G 5-CCA CGA ACG GAT ATG CGG G-3 19bp,
Downstream primer R2 5-CTG GGA ATG GGA CAA GCA CG-3 20bp.
Be the amount of water of 100 μ mol/L (being 100pmol/ μ l) concentration by every OD primer dilution before using, opened under the rotating speed that is preferably in 3000-4000 rev/min before the bottle cap dissolving centrifugal 1 minute that primer scatters and disappears when preventing to uncap.Before the use, experimentize after strong solution is diluted to working fluid 10pmol/ml.
(2) DNA extraction agent:
(3) pre-mixed PCR reaction solution
The premix reaction solution comprises 4 kinds, and every kind includes a pair of upstream and downstream amplimer, each corresponding genotype.Press the 1ml proportional quantity and calculate, the common composition is formulated as: the dNTP of 100 μ M, 20 μ l; 500U/ μ l Taq DNA Polymerase20 μ l; 2 * Quant One Step SYBR, 1300 μ l; The upstream primer 50 μ l of 10 μ M; The downstream primer 50 μ l of 10 μ M; RNase-free ddH
2O 860 μ l.
1 pair of primer of pipe 1 is:
Upstream primer F1A 5-GAA GCC CCG CAA ACC GA-3 17bp,
Downstream primer R1 5-CAG TGC ACA GCC TGA ACA GC-3 20bp;
1 pair of primer of pipe 2 is:
Upstream primer F1G 5-GAA GCC CCG CAA ACC GG-3 17bp,
Downstream primer R1 5-CAG TGC ACA GCC TGA ACA GC-3 20bp;
1 pair of primer of pipe 3 is:
Upstream primer F2A 5-CCA CGA ACG GAT ATG CGG A-3 19bp,
Downstream primer R2 5-CTG GGA ATG GGA CAA GCA CG-3 20bp;
1 pair of primer of pipe 4 is:
Upstream primer F2G 5-CCA CGA ACG GAT ATG CGG G-3 19bp,
Downstream primer R2 5-CTG GGA ATG GGA CAA GCA CG-3 20bp.
(4) standard positive control:
Standard positive template is to contain to contain the Puc18-T recombinant plasmid that mutation nucleotide fragment constitutes in two genes of OXTR-1 and OXTR-2, this reorganization material transformed into escherichia coli DH5 α propagation back alkaline lysis method of extracting, through DNA purification kit purifying, quantitatively and be diluted to 10 with spectrophotometric instrumentation A260
9Copy/ μ l ,-20 ℃ of preservations.Storage saves as 10
9Copy/ μ l, working concentration are 10
5Copy/ μ l.
Standard positive template provides 4 kinds, and every kind of concentration is 10
5Copy/ul.Sequence is as follows:
SEQ1：
TTGTGTGGGC?TGCTTTGTGG?CACAACTCCA?GGGGATGCCT?TTTACCTCAT
GGTCTATGAA?CAGGGTGTTC?CTAGGAGCTT?TGCAGTGCAC?AGCCTGAACA
GCTGAACATG?GCAGCCTCAT?CCAGTGCCCC?TTTCAGGAAA?CCATCCCTGT
TTTCTCAGTT?TGCGGGGCTT?CTTCCTCTGA?ATGCTAAGTC?AT；
SEQ2：
ATGACTTAGC?ATTCAGAGGA?AGAAGCCCCG?CAAACTGGGA?AAACAGGGAT
GGTTTCCTGA?AAGGGGCACT?GGATGAGGCT?GCCATGTTCA?GCTGTTCAGG
CTGTGCACTG?CAAAGCTCCT?AGGAACACCC?TGTTCATAGA?CCATGAGGTA
AAAGGCATCC?CCTGGAGTTG?TGCCACAAAG?CAGCCCACAC?AA；
SEQ3：
GGCTCCACTC?CTGGAGACTC?CACGAACGGA?TATGCTGAGT?CCACCGTGAA
ACAAACCGGG?AGGGCCGTGA?GGAGACCGCC?GCGTTTCTCT?TCAGACGCGG
GTAGGGCGTG?CTTGTCCCAT?TCCCAGGAAC?CCAACTCATC?TGAAACAACA
GGGCACAACC?GCCGGCCTCG?GGTTGCTCAG?CCGCCACCCC?AG；
SEQ4：
CTGGGGTGGC?GGCTGAGCAA?CCCGAGGCCG?GCGGTTGTGC?CCTGTTGTTT
CAGATGAGTT?GGGTTCCTGG?GAATGGGACA?AGCACGCCTT?ACCCGCGTCT
GAAGAGAAAC?GCGGCGGTCT?CCTCACGGCC?CTCCCGGTTT?GTTTCACGGT
GGACCCAGCA?TATCCGTTCG?TGGAGTCTCC?AGGAGAGGAG?CC。
(5) standard negative control:
Standard negative template is to contain the Puc18-T recombinant plasmid that 180 Nucleotide of arabidopsis gene segment constitute.Alkaline lysis method of extracting use in this reorganization material transformed into escherichia coli DH5 α propagation back, and is through DNA purification kit purifying, quantitative and be diluted to 10 with spectrophotometric instrumentation AA260
9Copy/ μ l ,-20 ℃ of preservations.Storage saves as 10
9Copy/ μ l, working concentration are 10
5Copy/ μ l.Sequence is as follows:
SEQ5：
TTCGTTATTG?AATCTTGTTT?TGGTTCTCTT?GTGTGGAATG?CATTGTTCTT
GCTTCTCTGG?AACTTGGGAT?ATGATGACAA?AAATACGATA?TCTCTAAGGC
TAATTGCTGT?GCGGATGAAA?TATATTTGCA?CGTCAGTCTG?ATCCCATTAT
GTATATAACA?TTAGGAGTTG?GTGCATGCTC。
Embodiment 2: the use of test kit
(1) preparation of sample
Sampling mode: use cotton swab wiping 10 times in cheek.
Attention: do not polluted in order to guarantee sample, take a sample and to take food and to drink water in preceding 30 minutes by food or beverage.
A) handle material: cotton swab transposition that will wiping in cheek is cut the cotton swab part with scissors in the 2ml centrifuge tube from its bar, add 400 μ l damping fluid GA.
B) add 20 μ l Proteinase K solution, 10 seconds mixings of vortex were placed 60 minutes for 56 ℃, and per therebetween 15 minutes vortex mixings for several times.
C) add 400 μ l damping fluid GB, fully put upside down mixing, placed 10 minutes for 70 ℃, this moment, the solution strain was limpid, and is brief centrifugal to remove the drop of cap wall.
D) add 200 μ l dehydrated alcohols, fully put upside down mixing, brief centrifugal to remove the drop of cap wall.
E) previous step gained solution and flocks are all added (adsorption column CR puts into collection tube) among the adsorption column CR, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.
F) in adsorption column CR, add 500 μ l damping fluid GD, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.
G) in adsorption column CR, add 700 μ l rinsing liquid PW, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back to collection tube with adsorption column.
H) in adsorption column CR, add 500 μ l rinsing liquid PW, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube.
I) adsorption column CR is put back in the collection tube, 12,000rpm (～13,400 * g) centrifugal 2 minutes, outwell waste liquid.Adsorption column CR room temperature was placed several minutes, thoroughly to dry rinsing remaining in the sorbing material.
J) adsorption column CR is changed in the clean centrifuge tube, to the unsettled dropping in adsorption film mid-way 20-50 μ l elution buffer TB, room temperature was placed 2-5 minute, and 12,000rpm (～13,400 * g) centrifugal 2 minutes.Repeat once.
(2) experimentation
A) getting the every pipe 23 μ l of pre-mixed PCR reaction solution by sample number n (sample number=number of awaiting test sample+1+positive control of negative control 1) is sub-packed in the reaction tubes.
B) the above-mentioned sample to be tested of handling well and feminine gender, positive control (must vibrate the mixing several seconds) are respectively got 2ul and added respectively in the reaction tubes, mixing, carries out pcr amplification reaction immediately at the low-speed centrifugal several seconds.
C) PCR condition
95℃10sec
(95 ℃ of 15sec, 60 ℃ of 60sec) * 40 circulations
(3) interpretation of result:
A) judging criterion of detection by quantitative SNP:
The absolute value of the difference of the Ct value of two curves is designated as | Δ Ct|, when | Δ Ct|≤1 is judged as heterozygous; When | Δ Ct| 〉=5 are judged as homozygous.
As figure, among Fig. 1 | Δ Ct| 〉=5, represent that this sample is the homozygous sample of OXTR-1 gene GG.Among Fig. 2, | Δ Ct| 〉=5, represent that this sample is the homozygous sample of OXTR-1 gene A A.Among Fig. 3, | Δ Ct|≤1, represent that this sample is an OXTR-1 Gene A heterozygous sample.Among Fig. 4 | Δ Ct| 〉=5, represent that this sample is the homozygous sample of OXTR-2 gene A A.Among Fig. 5, | Δ Ct|≤1, represent that this sample is an OXTR-2 Gene A heterozygous sample.Among Fig. 6, | Δ Ct| 〉=5, represent that this sample is the homozygous sample of OXTR-2 gene GG.
B) interpretation of result:
Sequence table
＜110〉Shanghai Yulong Biological Technology Co., Ltd
＜120〉a kind of kit for detecting susceptibility genes of autism of children
<160>11
<210>1
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to detect examination as susceptibility genes of autism of children
Detect the dna primer of the relevant sudden change of OXTR-1 gene order in the agent box, called after upstream primer F1A.
<400>1
gaagccccgc?aaaccga 17
<210>2
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to detect examination as susceptibility genes of autism of children
Detect the dna primer of the relevant sudden change of OXTR-1 gene order in the agent box, called after upstream primer F1G.
<400>2
gaagccccgc?aaaccgg 17
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to detect examination as susceptibility genes of autism of children
Detect the dna primer of the relevant sudden change of OXTR-1 gene order in the agent box, called after downstream primer R1.
<400>3
cagtgcacag?cctgaacagc 20
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to detect examination as susceptibility genes of autism of children
Detect the dna primer of the relevant sudden change of OXTR-2 gene order in the agent box, called after upstream primer F2A.
<400>4
ccacgaacgg?atatgcgga 19
<210>5
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to detect examination as susceptibility genes of autism of children
Detect the dna primer of the relevant sudden change of OXTR-2 gene order in the agent box, called after upstream primer F2G.
<400>5
ccacgaacgg?atatgcggg 19
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to detect examination as susceptibility genes of autism of children
Detect the dna primer of the relevant sudden change of OXTR-2 gene order in the agent box, called after downstream primer R2.
<400>6
ctgggaatgg?gacaagcacg 20
<210>7
<211>192
<212>DNA
＜213〉Genus Homo (Homo)
<400>7
ttgtgtgggc?tgctttgtgg?cacaactcca?ggggatgcct?tttacctcat?ggtctatgaa 60
cagggtgttc?ctaggagctt?tgcagtgcac?agcctgaaca?gctgaacatg?gcagcctcat 120
ccagtgcccc?tttcaggaaa?ccatccctgt?tttctcagtt?tgcggggctt?cttcctctga 180
atgctaagtc?at 192
<210>8
<211>192
<212>DNA
＜213〉Genus Homo (Homo)
<400>8
atgacttagc?attcagagga?agaagccccg?caaactggga?aaacagggat?ggtttcctga 60
aaggggcact?ggatgaggct?gccatgttca?gctgttcagg?ctgtgcactg?caaagctcct 120
aggaacaccc?tgttcataga?ccatgaggta?aaaggcatcc?cctggagttg?tgccacaaag 180
cagcccacac?aa 192
<210>9
<211>192
<212>DNA
＜213〉Genus Homo (Homo)
<400>9
ggctccactc?ctggagactc?cacgaacgga?tatgctgagt?ccaccgtgaa?acaaaccggg 60
agggccgtga?ggagaccgcc?gcgtttctct?tcagacgcgg?gtagggcgtg?cttgtcccat 120
tcccaggaac?ccaactcatc?tgaaacaaca?gggcacaacc?gccggcctcg?ggttgctcag 180
ccgccacccc?ag 192
<210>10
<211>192
<212>DNA
＜213〉Genus Homo (Homo)
<400>10
ctggggtggc?ggctgagcaa?cccgaggccg?gcggttgtgc?cctgttgttt?cagatgagtt 60
gggttcctgg?gaatgggaca?agcacgcctt?acccgcgtct?gaagagaaac?gcggcggtct 120
cctcacggcc?ctcccggttt?gtttcacggt?ggacccagca?tatccgttcg?tggagtctcc 180
aggagaggag?cc 192
<210>11
<211>180
<212>DNA
＜213〉Arabidopis thaliana (Arabidopsis thaliana)
<400>11
ttcgttattg?aatcttgttt?tggttctctt?gtgtggaatg?cattgttctt?gcttctctgg 60
aacttgggat?atgatgacaa?aaatacgata?tctctaaggc?taattgctgt?gcggatgaaa 120
tatatttgca?cgtcagtctg?atcccattat?gtatataaca?ttaggagttg?gtgcatgctc 180
Claims (3)
1. kit for detecting susceptibility genes of autism of children is characterized in that: by the DNA extraction agent, and pre-mixed PCR reaction solution, standard positive reference substance, standard negative reference substance is formed; Described pre-mixed PCR reaction solution contains SYBR green I, and the amplimer of OXTR-1 gene relevant with autism of children and OXTR-2 gene mutation site; Amplimer is upstream primer and the normal downstream primer that comprises the mutational site, and sequence is as follows:
The primer of amplification gene OXTR-1:
Upstream primer F1A 5-GAA GCC CCG CAA ACC GA-3 17bp,
Upstream primer F1G 5-GAA GCC CCG CAA ACC GG-3 17bp,
Downstream primer R1 5-CAG TGC ACA GCC TGA ACA GC-3 20bp;
The primer of amplification gene OXTR-2:
Upstream primer F2A 5-CCA CGAACG GAT ATG CGG A-3 19bp,
Upstream primer F2G 5-CCA CGAACG GAT ATG CGG G-3 19bp,
Downstream primer R2 5-CTG GGAATG GGA CAA GCA CG-3 20bp.
2. kit for detecting susceptibility genes of autism of children as claimed in claim 1 is characterized in that: described positive reference substance has 4, comprises following sequence respectively:
SEQ1：
TTGTGTGGGC TGCTTTGTGG CACAACTCCA GGGGATGCCT
TTTACCTCAT GGTCTATGAA CAGGGTGTTC CTAGGAGCTT
TGCAGTGCAC AGCCTGAACA GCTGAACATG GCAGCCTCAT
CCAGTGCCCC TTTCAGGAAA CCATCCCTGT TTTCTCAGTT
TGCGGGGCTT CTTCCTCTGA ATGCTAAGTC AT；
SEQ?2：
ATGACTTAGC ATTCAGAGGA AGAAGCCCCG CAAACTGGGA
AAACAGGGAT GGTTTCCTGA AAGGGGCACT GGATGAGGCT
GCCATGTTCA GCTGTTCAGG CTGTGCACTG CAAAGCTCCT
AGGAACACCC TGTTCATAGA CCATGAGGTA AAAGGCATCC
CCTGGAGTTG TGCCACAAAG CAGCCCACAC AA；
SEQ3：
GGCTCCACTC CTGGAGACTC CACGAACGGA TATGCTGAGT
CCACCGTGAA ACAAACCGGG AGGGCCGTGA GGAGACCGCC
GCGTTTCTCT TCAGACGCGG GTAGGGCGTG CTTGTCCCAT
TCCCAGGAAC CCAACTCATC TGAAACAACA GGGCACAACC
GCCGGCCTCG GGTTGCTCAG CCGCCACCCC AG；
SEQ4：
CTGGGGTGGC GGCTGAGCAA CCCGAGGCCG GCGGTTGTGC
CCTGTTGTTT CAGATGAGTT GGGTTCCTGG GAATGGGACA
AGCACGCCTT ACCCGCGTCT GAAGAGAAAC GCGGCGGTCT
CCTCACGGCC CTCCCGGTTT GTTTCACGGT GGACCCAGCA
TATCCGTTCG TGGAGTCTCC AGGAGAGGAG CC。
3. kit for detecting susceptibility genes of autism of children as claimed in claim 1 or 2 is characterized in that: the sequence that described negative control product comprise is:
SEQ5：TTCGTTATTG AATCTTGTTT TGGTTCTCTT GTGTGGAATG
CATTGTTCTT GCTTCTCTGG AACTTGGGAT ATGATGACAA
AAATACGATA TCTCTAAGGC TAATTGCTGT GCGGATGAAA
TATATTTGCA CGTCAGTCTG ATCCCATTAT GTATATAACA
TTAGGAGTTG GTGCATGCTC。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810200630A CN101684496A (en) | 2008-09-27 | 2008-09-27 | Kit for detecting susceptibility genes of autism of children |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810200630A CN101684496A (en) | 2008-09-27 | 2008-09-27 | Kit for detecting susceptibility genes of autism of children |
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| CN101684496A true CN101684496A (en) | 2010-03-31 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200810200630A Pending CN101684496A (en) | 2008-09-27 | 2008-09-27 | Kit for detecting susceptibility genes of autism of children |
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| CN (1) | CN101684496A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290135A (en) * | 2013-06-26 | 2013-09-11 | 北京迈基诺基因科技有限责任公司 | Screening method of infantile autism gene |
| CN113057969A (en) * | 2021-03-09 | 2021-07-02 | 吴志新 | Clinical application experiment method of mesenchymal stem cells to infantile autism |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007812A2 (en) * | 2003-07-03 | 2005-01-27 | University Of Medicine And Dentistry Of New Jersey | Genes as diagnostic tools for autism |
| US20060141519A1 (en) * | 2003-07-03 | 2006-06-29 | Millonig James H | Compositions and methods for diagnosing autism |
| WO2006087634A2 (en) * | 2005-02-17 | 2006-08-24 | Integragen | Uses of human autism susceptibility gene encoding a kinase |
-
2008
- 2008-09-27 CN CN200810200630A patent/CN101684496A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007812A2 (en) * | 2003-07-03 | 2005-01-27 | University Of Medicine And Dentistry Of New Jersey | Genes as diagnostic tools for autism |
| US20060141519A1 (en) * | 2003-07-03 | 2006-06-29 | Millonig James H | Compositions and methods for diagnosing autism |
| WO2006087634A2 (en) * | 2005-02-17 | 2006-08-24 | Integragen | Uses of human autism susceptibility gene encoding a kinase |
Non-Patent Citations (2)
| Title |
|---|
| E LERER ET AL.: "Association between the oxytocin receptor (OXTR) gene and autism: relationship to Vineland Adaptive Behavior Scales and cognition", 《MOLECULAR PSYCHIATRY》 * |
| SUPING WU ET AL.: "Positive Association of the Oxytocin Receptor Gene (OXTR) with Autism in the Chinese Han population", 《BIOL PSYCHIATRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290135A (en) * | 2013-06-26 | 2013-09-11 | 北京迈基诺基因科技有限责任公司 | Screening method of infantile autism gene |
| CN113057969A (en) * | 2021-03-09 | 2021-07-02 | 吴志新 | Clinical application experiment method of mesenchymal stem cells to infantile autism |
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Application publication date: 20100331 |