CN104673893A - Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof - Google Patents
Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular cytogenetics and relates to an amplified composition for fast gene detection of microdeletion of Y chromosomes, a kit containing the same and an application thereof. The amplified composition comprises a group A and a group B, wherein each group comprises a pair of primers respectively in AZFa, AZFb and AZFc areas and two pairs of internal control primers ZFY and SRY; the primers in the group A have the nucleotide sequence shown in SEQ ID No.1-1O; the primers in the group B have the nucleotide sequence shown in SEQ ID No.11-2O. The amplified composition, the kit and the application have the advantages that on the basis of the prior art, multiple PCR (Polymerase Chain Reaction) buffering liquid and Tag polymerase are improved, and trace whole-blood amplification is directly adopted due to less blood consumption, so that the pain of a patient is reduced; and the procedure of extracting DNA is omitted, so that the time and the labor are saved and the economic expenditure is greatly reduced.
Description
Technical field
The invention belongs to cellular elements genetic arts, relate to the micro-deleted Amplification thing of rapid gene detection of a kind of Y chromosome, the test kit containing this Amplification thing and application thereof.
Background technology
Y chromosome is micro-deleted is that the male sex is without major reason that is smart or the few weak essence of severe.Cause in the patient of male sterility at spermatogenesis obstacle, the micro-deleted incidence of Y chromosome occupy the second inherited genetic factors, is only second to kirschner syndromes.Based on the importance of the micro-deleted detection of Y chromosome, European andrology can and European molecular genetic quality net combine and be proposed the micro-deleted inspection guide of Y chromosome, become a standardized inspection item.Current andrology requires that the micro-deleted gene test of Y chromosome is as the routine inspection of male sterility, can find out the cause of disease on the one hand, on the other hand for the diagnosis of male sterility patient clinical provides foundation and guidance for the male sterility patient of the not clear cause of disease.
Tiepolo in 1976 etc. find that in 1170 routine male sterility patients 6 examples find deletion of long arm under the microscope without sperm patient, infer to there is Gene of Spermatogenesis on Y chromosome is long-armed, be referred to as without sperm zone (azoospermia factor region AZF).On this basis, AZF Further Division is AZFa, AZFb and AZFc tri-subprovinces by Vogt in 1996.Disappearance or the sudden change in AZF district may cause oligospermatism or azoospermia.
2004, in the micro-deleted inspection guide of Y chromosome that release combined by Europe andrology meeting and European molecular genetic quality net, be recommended in each AZF region and use 2 sequence tagged site: SY84 (AZFa), SY86 (AZFa), SY127 (AZFb), SY134 (AZFb), SY254 (AZFc), SY255 (AZFc), these 6 sequence tagged sites can detect the AZF disappearance more than 95%.Because guide is strict, specification, high performance reproducibility, it is the micro-deleted detection method of Y chromosome generally followed in the world at present.This method of inspection extracting peripheral blood DNA, carries out multi-PRC reaction by 5 pairs of primers.The micro-deleted of male Y chromosome whether is there is finally by electrophoresis detection.
For a long time, it is almost round pcr " iron rule " that DNA must be adopted to carry out pcr amplification.For effectively increasing to various " natural sample ", some world-famous biotech companies such as Quiagen, Promega etc. competitively develop the DNA extraction technique of efficient, are used for downstream PCR reaction with the DNA of the high-quality by extracting.But carrying out DNA extracting self has certain shortcoming.Comprise: in (1) extractive process, there is consume.No matter how efficiently DNA extraction technique, all can not by the DNA in sample without consume extracting process.Special all the more so when micro-example extracting.(2) bothersome effort is operated.Even if DNA extracting the most efficiently, usually consuming time also at 45 minutes to 1 hours.When sample size is larger, extracting DNA can occupy the considerable time and efforts of experimenter.(3) increase economic expenditure, DNA extraction agent is expensive efficiently.Cost occupied by extracting DNA reacts self higher than PCR even far away.(4) even if carry out DNA extracting, in the sample that small part is special, be also difficult to remove PCR response inhabitation thing.
Chinese patent 200710074317.X discloses the micro-deleted detection kit of male Y chromosome and detection method, 200910094618.8 disclose the gene tester that a kind of Y chromosome is micro-deleted, what all adopt is from peripheral blood, extract the method that DNA utilizes multiplexed PCR amplification, operation is wasted time and energy, and increases economic expenditure.
Summary of the invention
The invention provides a kind of can fast, stable, efficient and directly to the Amplification thing that the micro-deleted rapid gene of the Y chromosome of the multiple gene fragment of the disposable amplification of whole blood detects.
The present invention also provides a kind of test kit containing this Amplification thing.
The technical solution adopted for the present invention to solve the technical problems is:
The Amplification thing that the micro-deleted rapid gene of Y chromosome detects, this Amplification thing comprises AB two groups, and often group comprises each pair of primers in AZFa, AZFb, AZFc region and two couples of internal reference primer ZFY and SRY, and the combination of primers often in group is as follows:
A group:
Internal reference ZFY primer pair, it has the nucleotide sequence as shown in SEQ ID No.1 and SEQ ID No.2;
ZFY-F：5’-ACC RCT GTA CTG ACT GTG ATT ACA C -3’
ZFY-R：5’-GCA CYT CTT TGG TAT CYG AGA AAG T -3’
Internal reference SRY primer pair, it has the nucleotide sequence as shown in SEQ ID No.3 and SEQ ID No.4;
SRY-F：5’-GAA TAT TCC CGC TCT CCG GA -3’
SRY-R：5’-GCT GGT GCT CCA TTC TTG AG -3’
SY254 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.5 and SEQ ID No.6;
SY254-F:5’-GGG TGT TAC CAG AAG GCA AA -3’
SY254-R:5’-GAA CCG TAT CTA CCA AAG CAG C -3’
SY86 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.7 and SEQ ID No.8;
SY86-F：5’-GTG ACA CAC AGA CTA TGC TTC -3’
SY86-R：5’-ACA CAC AGA GGG ACA ACC CT -3’
SY127 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.9 and SEQ ID No.10;
SY127-F：5’-GGC TCA CAA ACG AAA AGA AA -3’
SY127-R：5’-CTG CAG GCA GTA ATA AGG GA -3’
B group: internal reference ZFY primer pair, it has the nucleotide sequence as shown in SEQ ID No.11 and SEQ ID No.12;
ZFY-F：5’-ACC RCT GTA CTG ACT GTG ATT ACA C -3’
ZFY-R：5’-GCA CYT CTT TGG TAT CYG AGA AAG T -3’
Internal reference SRY primer pair, it has the nucleotide sequence as shown in SEQ ID No.13 and SEQ ID No.14;
SRY-F：5’-GAA TAT TCC CGC TCT CCG GA -3’
SRY-R：5’-GCT GGT GCT CCA TTC TTG AG -3’
SY84 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.15 and SEQ ID No.16;
SY84-F：5’-AGA AGG GTC TGA AAG CAG GT -3’
SY84-R：5’-GCC TAC TAC CTG GAG GCT TC -3’
SY134 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.17 and SEQ ID No.18;
SY134-F：5’-GTC TGC CTC ACC ATA AAA CG -3’
SY134-R：5’-ACC ACT GCC AAA ACT TTC AA -3’
SY255 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.19 and SEQ ID No.20;
SY255-F：5’-GTT ACA GGA TTC GGC GTG AT -3’
SY255-R：5’-CTC GTC ATG TGC AGC CAC -3’。
The micro-deleted detection kit of a kind of rapid detection male Y chromosome, described test kit comprises: Amplification thing described in claim 1, PCR reaction buffer, dNTPs, archaeal dna polymerase.Test kit of the present invention can be used for detecting simultaneously and comprises Y chromosome AZFa subprovince sY84 and sY86 site, Y chromosome AZFb subprovince sY127 and sY134 site, the genetically deficient in Y chromosome AZFc subprovince sY254 and sY255 site.
As preferably, PCR reaction buffer comprises 300mM Ttricine, 25mM (NH
4)
2sO
4, 17.5mM MgCl
2, 30% glycerine.PCR react buffer system need to increase one or more can effectively with the combination of template, reduce the impact of template DNA " GC " secondary structure, increase the additive of PCR atopic.
As preferably, the archaeal dna polymerase of employing is saltant type Tag enzyme Hemo Klen Tag (Niu Yinglun biotechnology (Beijing) company limited).The archaeal dna polymerase selected can tolerate inhibition contained in whole blood.
As preferably, dNTPs concentration is 2.5mM.
Utilize the method that described test kit rapid detection male Y chromosome is micro-deleted, described method mainly comprises the following steps: (1) peripheral blood collection: extract peripheral blood 1ml mixing with anticoagulant heparin pipe for subsequent use; (2) direct peripheral blood carries out multiplexed PCR amplification as substrate, carries out two groups of multi-PRC reactions, often overlaps reaction system and carries out multi-PRC reaction by Amplification thing according to claim 1; (3) agarose gel electrophoresis detects PCR primer: get 10 μ l PCR primer, and after TAE dyeing, through 1.5% agarose gel electrophoresis, electrophoretic voltage 120V, electrophoresis time 90 minutes, by gel imaging instrument observation analysis.
The method, without the need to extracting blood DNA, directly uses 5 μ l peripheral bloods as amplification substrate, improves conventional multiplex PCR system, carries out the powerful multi-PRC reaction of two covers, and often overlap reaction system and carry out multi-PRC reaction by 5 pairs of primers, increase 5 objective fragments; (3) get 10 μ l PCR primer, agarose gel electrophoresis detects.Adopt the method for the invention to detect male Y chromosome missing gene, have easy fast, efficient, accurately, the advantage such as low cost.
As preferably, the PCR reaction system of 25 μ l comprises: PCR reaction buffer: 300mM Ttricine, 25mM (NH
4)
2sO
4, 17.5mM MgCl
2, 30% glycerine, 5 μ l; Whole blood 2.5 μ l; 2 μ l 2.5mMdNTPs; 1 μ l 100 units/μ l saltant type Tag enzyme Hemo Klen Tag (Niu Yinglun biotechnology (Beijing) company limited); 1.25-2.5 μM mixed assemblage primer 2 .5 μ l; 12 μ l H
2o.
As preferably, PCR cycling condition is: 95 DEG C of initial denaturation 5min, 95 DEG C of sex change 30s, 58 DEG C of annealing 90s, and 72 DEG C extend 60s, and after 35 reaction cycle, 72 DEG C extend 15min, preserves samples for 4 DEG C and uses in order to next step experiment.
The invention has the beneficial effects as follows: the present invention, in prior art, improves multiplex PCR damping fluid and Tag polysaccharase, directly adopt micro whole blood amplification, because its blood using amount is few, reduce the misery of patient; Remove extracting DNA program from, time saving and energy saving, and greatly reduce economic expenditure.
Accompanying drawing explanation
Fig. 1 is Y chromosome micro-deleted detection negative findings electrophorogram;
Fig. 2 is the electrophorogram of the micro-deleted gene test result of Y chromosome;
In Fig. 1 and Fig. 2, be followed successively by duct 1 from left to right for DL500 DNA marker, duct 2 is blank, duct 3 is women's whole blood control, duct 4 (A group), 5 (B groups) are normal male whole blood control, duct 6 (A group), 7 (B groups) are for detecting sample, and ordinate zou is DNA molecular amount.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation make the present invention and/or change all will fall into scope.
In the present invention, if not refer in particular to, all parts, per-cent are weight unit, and the equipment adopted and raw material etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the ordinary method of this area.
Embodiment 1:
The method that the micro-deleted rapid gene of Y chromosome detects, concrete grammar is as follows:
1, peripheral blood collection: extract peripheral blood 1ml mixing with anticoagulant heparin pipe for subsequent use;
2, multiplex PCR system amplification multiplex PCR divides A group and B group two cover system to carry out
A group PCR reaction system: the PCR reaction system of 25 μ l is: PCR cushions buffer:300mM Ttricine, 25mM (NH
4)
2sO
4, 17.5mM MgCl
2, 30% glycerine, 5 μ l; 1X mix primer A 2.5 μ l; Whole blood 2.5 μ l; 2 μ l 2.5mM dNTPs (Takara); 1 μ l 100 units/μ l saltant type Tag enzyme (Niu Yinglun biotechnology (Beijing) company limited); ddH
2o 12 μ l.
The preparation of A group 5X mix primer (mix primer A): all synthetic primers often pipe content are 1OD, ZFY-F 3.7nmol/OD, ZFY-R 3.6nmol/OD, SRY-F 4.8nmol/OD, SRY-R 4.9nmol/OD, SY254-F 4.2nmol/OD, SY254-R 4.1nmol/OD, SY86-F 4.3nmol/OD, SY86-R 4.3nmol/OD, SY127-F 3.9nmol/OD, SY127-R 4.2nmol/OD.Get 1 pipe ZFY-F and add 250 μ l deionized waters, mixing, liquid is all transferred to ZFY-R pipe, mixing, repeat step, shifting SRY-F, SRY-R, SY254-F, SY254-R, SY86-F, SY86-R, SY127-F and SY127-R successively is all dissolved in 250 μ l deionized waters, packing, dated primer information.
The each primer sequence of A group:
Internal reference ZFY primer pair:
ZFY-F：5’-ACC RCT GTA CTG ACT GTG ATT ACA C -3’
ZFY-R：5’-GCA CYT CTT TGG TAT CYG AGA AAG T -3’
Internal reference SRY primer pair:
SRY-F：5’-GAA TAT TCC CGC TCT CCG GA -3’
SRY-R：5’-GCT GGT GCT CCA TTC TTG AG -3’
SY254 site primer pair:
SY254-F:5’-GGG TGT TAC CAG AAG GCA AA -3’
SY254-R:5’-GAA CCG TAT CTA CCA AAG CAG C -3’
SY86 site primer pair:
SY86-F：5’-GTG ACA CAC AGA CTA TGC TTC -3’
SY86-R：5’-ACA CAC AGA GGG ACA ACC CT -3’
SY127 site primer pair:
SY127-F：5’-GGC TCA CAA ACG AAA AGA AA -3’
SY127-R：5’-CTG CAG GCA GTA ATA AGG GA -3’。
B group PCR reaction system: with A tube reaction system, wherein primer is: 1X mix primer B2.5 μ l.
The preparation of B group 5X mix primer (mix primer B): all synthetic primers often pipe content are 1OD, ZFY-F 3.7nmol/OD, ZFY-R 3.6nmol/OD, SRY-F 4.8nmol/OD, SRY-R 4.9nmol/OD, SY84-F 4.1nmol/OD, SY84-R 4.9nmol/OD, SY134-F 4.6nmol/OD, SY134-R 4.5nmol/OD, SY255-F 4.5nmol/OD, SY255-R 5.5nmol/OD.Method, with the preparation of A pipe 5X mix primer, makes ZFY-F, ZFY-R, SRY-F, SRY-R, SY84-F, SY84-R, SY134-F, SY134-R, SY255-F and SY255-R all be dissolved in 250 μ l deionized waters, packing, dated primer information.
The each primer sequence of B group:
Internal reference ZFY primer pair:
ZFY-F：5’-ACC RCT GTA CTG ACT GTG ATT ACA C -3’
ZFY-R：5’-GCA CYT CTT TGG TAT CYG AGA AAG T -3’
Internal reference SRY primer pair:
SRY-F：5’-GAA TAT TCC CGC TCT CCG GA -3’
SRY-R：5’-GCT GGT GCT CCA TTC TTG AG -3’
SY84 site primer pair:
SY84-F：5’-AGA AGG GTC TGA AAG CAG GT -3’
SY84-R：5’-GCC TAC TAC CTG GAG GCT TC -3’
SY134 site primer pair:
SY134-F：5’-GTC TGC CTC ACC ATA AAA CG -3’
SY134-R：5’-ACC ACT GCC AAA ACT TTC AA -3’
SY255 site primer pair:
SY255-F：5’-GTT ACA GGA TTC GGC GTG AT -3’
SY255-R：5’-CTC GTC ATG TGC AGC CAC -3’。
Carry out thermal cycle reaction by following temperature: 95 DEG C of initial denaturation 5min, 95 DEG C of sex change 30s, 58 DEG C of annealing 90s, 72 DEG C extend 60s, and after 35 reaction cycle, 72 DEG C extend 15min, preserve sample for 4 DEG C and use in order to next step experiment.
3, agarose gel electrophoresis
Prepare 1.5% gel: take 0.75g agar Icing Sugar and be placed in Erlenmeyer flask, add 50ml 1X TAE, bottleneck back-off small beaker, microwave-oven-heating boils 3 times, melts completely to agarose, adds vegetable dye GelRedTm (BIOTUIM) 3 μ l, again boil in microwave oven, shake up.
Prepared by offset plate: get organic glass box inside groove wash clean in electrophoresis chamber, dry, put into glue sheet glass and seal sheet glass and inner slot end, form mould, inside groove is placed in level attitude, and puts comb well in fixed position, treat that agarose is cooled to 65 DEG C, mixing is poured on inside groove sheet glass carefully, glue is slowly launched, until whole glass-board surface forms even glue-line, left at room temperature treats that gel solidifies completely, vertically gently pull out comb, till interpolation 1X TAE damping fluid did not have glue 1-2mm.
Application of sample: mix 5 μ l PCR primer and 1 μ l 6X sample-loading buffer on point template, respectively sample is added in the sample cell of offset plate with 10 μ l micropipets;
Electrophoresis: voltage 100V, electrophoresis time 90min;
Observe and take a picture: BIO-RAD gel imaging instrument is taken pictures preservation;
4, result judges and optimizes:
Electrophoresis reaction product pillar location is as follows: ZFY 495bp, SRY472bp, SY254 400bp, SY86 320bp, SY127 274bp, SY84 326bp, SY134 301bp, SY255 126bp.
SRY determines gene as male gender, in an experiment as internal reference, ZFY is positioned on Y chromosome galianconism and X chromosome, also as internal reference, usual male sex's sample all should amplify internal reference contrast band, if testing sample is without this band, illustrate that PCR primer fails amplification, should re-start whole blood PCR.Women's sample can only amplify ZFY mono-band, uses women whole blood to be used for specificity and the pollution condition of monitoring experiment as outer contrast, and whether water to be used in monitoring experiment process reagent etc. as blank has pollution.
AZFc is clinical modal deletion type.If single disappearance appears in SY254 or SY255 in experiment, normally caused by experimental error, as unreasonable in reagent design or Saving specimen etc. has problem; AZFa occurs that Single locus disappearance is also very rare, and after getting rid of experimental implementation error, judge that AZFa region segments lacks, conclusion must carefully be checked before judging; AZFb areas case roughly the same.
Fig. 1 is Y chromosome micro-deleted detection negative findings electrophorogram, be followed successively by duct 1 from left to right for DL500mark, duct 2 is blank, duct 3 is normal female contrast, duct 4 (A group), 5 (B groups) are normal male whole blood control, and duct 6 (A group), 7 (B groups) are for detecting sample.Visible AZFa, AZFb, AZFc district is showed no disappearance.
Fig. 2 is the electrophorogram of the micro-deleted gene test positive findings of Y chromosome, be followed successively by duct 1 from left to right for DL500mark, duct 2 is blank, duct 3 is female control, duct 4 (A group), 5 (B groups) are normal male whole blood control, and duct 6 (A group), 7 (B groups) are for detecting sample.SY254 disappearance in visible duct 6, SY255 disappearance in duct 7, the therefore micro-deleted detection of this sample Y chromosome, is shown in that AZFc district lacks.
Embodiment 2
For the test kit that rapid detection male Y chromosome is micro-deleted, comprising: PCR reaction buffer, dNTP, archaeal dna polymerase, mixing multiplex amplification composition A liquid and B liquid.
PCR damping fluid comprises 300mM Ttricine, 25mM (NH
4)
2sO
4, 17.5mM MgCl
2, 30% glycerine.
The preparation of 5X Amplification thing A liquid: all synthetic primers often pipe content are 1OD, ZFY-F 3.7nmol/OD, ZFY-R 3.6nmol/OD, SRY-F 4.8nmol/OD, SRY-R 4.9nmol/OD, SY254-F 4.2nmol/OD, SY254-R 4.1nmol/OD, SY86-F 4.3nmol/OD, SY86-R 4.3nmol/OD, SY127-F 3.9nmol/OD, SY127-R 4.2nmol/OD.Get 1 pipe ZFY-F and add 250 μ l deionized waters, mixing, liquid is all transferred to ZFY-R pipe, mixing, repeat step, shifting SRY-F, SRY-R, SY254-F, SY254-R, SY86-F, SY86-R, SY127-F and SY127-R successively is all dissolved in 250 μ l deionized waters, packing, dated primer information.
The each primer sequence of A liquid:
Internal reference ZFY primer pair:
ZFY-F：5’-ACC RCT GTA CTG ACT GTG ATT ACA C -3’
ZFY-R：5’-GCA CYT CTT TGG TAT CYG AGA AAG T -3’
Internal reference SRY primer pair:
SRY-F：5’-GAA TAT TCC CGC TCT CCG GA -3’
SRY-R：5’-GCT GGT GCT CCA TTC TTG AG -3’
SY254 site primer pair:
SY254-F:5’-GGG TGT TAC CAG AAG GCA AA -3’
SY254-R:5’-GAA CCG TAT CTA CCA AAG CAG C -3’
SY86 site primer pair:
SY86-F：5’-GTG ACA CAC AGA CTA TGC TTC -3’
SY86-R：5’-ACA CAC AGA GGG ACA ACC CT -3’
SY127 site primer pair:
SY127-F：5’-GGC TCA CAA ACG AAA AGA AA -3’
SY127-R：5’-CTG CAG GCA GTA ATA AGG GA -3’。
The preparation of 5X Amplification thing B liquid: all synthetic primers often pipe content are 1OD, ZFY-F 3.7nmol/OD, ZFY-R 3.6nmol/OD, SRY-F 4.8nmol/OD, SRY-R 4.9nmol/OD, SY84-F 4.1nmol/OD, SY84-R 4.9nmol/OD, SY134-F 4.6nmol/OD, SY134-R 4.5nmol/OD, SY255-F 4.5nmol/OD, SY255-R 5.5nmol/OD.Method, with the preparation of A pipe 5X mix primer, makes ZFY-F, ZFY-R, SRY-F, SRY-R, SY84-F, SY84-R, SY134-F, SY134-R, SY255-F and SY255-R all be dissolved in 250 μ l deionized waters, packing, dated primer information.
The each primer sequence of B group: internal reference ZFY primer pair:
ZFY-F：5’-ACC RCT GTA CTG ACT GTG ATT ACA C -3’
ZFY-R：5’-GCA CYT CTT TGG TAT CYG AGA AAG T -3’
Internal reference SRY primer pair:
SRY-F：5’-GAA TAT TCC CGC TCT CCG GA -3’
SRY-R：5’-GCT GGT GCT CCA TTC TTG AG -3’
SY84 site primer pair:
SY84-F：5’-AGA AGG GTC TGA AAG CAG GT -3’
SY84-R：5’-GCC TAC TAC CTG GAG GCT TC -3’
SY134 site primer pair:
SY134-F：5’-GTC TGC CTC ACC ATA AAA CG -3’
SY134-R：5’-ACC ACT GCC AAA ACT TTC AA -3’
SY255 site primer pair:
SY255-F：5’-GTT ACA GGA TTC GGC GTG AT -3’
SY255-R：5’-CTC GTC ATG TGC AGC CAC -3’。
Archaeal dna polymerase is saltant type Tag enzyme.
Embodiment 3 utilizes this test kit to detect the micro-deleted method of male Y chromosome
1, peripheral blood collection: extract peripheral blood 1ml mixing with anticoagulant heparin pipe for subsequent use;
2, multiplex PCR system amplification
Multiplex PCR divides A group and B group two cover system to carry out
10.A group PCR reaction system: the PCR reaction system of 25 μ l comprises: PCR reaction buffer: 300mM Ttricine, 25mM (NH
4)
2sO
4, 17.5mM MgCl
2, 30% glycerine, 5 μ l; Whole blood 2.5 μ l; 2 μ l 2.5mM dNTPs; 1 μ l 100 units/μ l saltant type Tag enzyme Hemo Klen Tag (Niu Yinglun biotechnology (Beijing) company limited); 1.25-2.5 μM mixed assemblage primer 2 .5 μ l; 12 μ l H
2o.
B group PCR reaction system: with A tube reaction system, wherein primer is: 1X mix primer B2.5 μ l.
A group and B group two cover system carry out thermal cycle reaction by following temperature: 95 DEG C of initial denaturation 5min, 95 DEG C of sex change 30s, 58 DEG C of annealing 90s, and 72 DEG C extend 60s, and after 35 reaction cycle, 72 DEG C extend 15min, preserve sample for 4 DEG C and use in order to next step experiment.
3, agarose gel electrophoresis
Prepare 1.5% gel: take 0.75g agar Icing Sugar and be placed in Erlenmeyer flask, add 50ml 1X TAE, bottleneck back-off small beaker, microwave-oven-heating boils 3 times, melts completely to agarose, adds vegetable dye GelRed
tm(BIOTUIM company) 3 μ l, again boils in microwave oven, shakes up.
Prepared by offset plate: get organic glass box inside groove wash clean in electrophoresis chamber, dry, put into glue sheet glass and seal sheet glass and inner slot end, form mould, inside groove is placed in level attitude, and puts comb well in fixed position, treat that agarose is cooled to 65 DEG C, mixing is poured on inside groove sheet glass carefully, glue is slowly launched, until whole glass-board surface forms even glue-line, left at room temperature treats that gel solidifies completely, vertically gently pull out comb, till interpolation 1X TAE damping fluid did not have glue 1-2mm.
Application of sample: mix 5 μ l PCR primer and sample adds in the sample cell of offset plate by 1 μ l 6X sample-loading buffer 10 μ l micropipets respectively on point template;
Electrophoresis: voltage 100V, electrophoresis time 90min;
Observe and take a picture: BIO-RAD gel imaging instrument is taken pictures preservation;
4, result judges and optimizes:
Electrophoresis reaction product pillar location is as follows: ZFY 495bp, SRY472bp, SY254 400bp, SY86 320bp, SY127 274bp, SY84 326bp, SY134 301bp, SY255 126bp.
SRY determines gene as male gender, in an experiment as internal reference, ZFY is positioned on Y chromosome galianconism and X chromosome, also as internal reference, usual male sex's sample all should amplify internal reference contrast band, if testing sample is without this band, illustrate that PCR primer fails amplification, should re-start whole blood PCR.Women's sample can only amplify ZFY mono-band, uses women whole blood to be used for specificity and the pollution condition of monitoring experiment as outer contrast, and whether water to be used in monitoring experiment process reagent etc. as blank has pollution.
AZFc is clinical modal deletion type.If single disappearance appears in SY254 or SY255 in experiment, normally caused by experimental error, as unreasonable in reagent design or Saving specimen etc. has problem; AZFa occurs that Single locus disappearance is also very rare, and after getting rid of experimental implementation error, judge that AZFa region segments lacks, conclusion must carefully be checked before judging; AZFb areas case roughly the same.
Fig. 1 is Y chromosome micro-deleted detection negative findings electrophorogram, be followed successively by duct 1 from left to right for DL500 DNA marker, duct 2 is blank, duct 3 is normal female contrast, duct 4 (A group), 5 (B groups) are normal male whole blood control, and duct 6 (A group), 7 (B groups) are for detecting sample.Visible AZFa, AZFb, AZFc district is showed no disappearance.
Fig. 2 is the electrophorogram of the micro-deleted gene test positive findings of Y chromosome, be followed successively by duct 1 from left to right for DL500 DNA marker, duct 2 is blank, duct 3 is normal female whole blood control, duct 4 (A group), 5 (B groups) are normal male whole blood control, and duct 6 (A group), 7 (B groups) are for detecting sample.SY254 disappearance in visible duct 6, SY255 disappearance in duct 7, the therefore micro-deleted detection of this sample Y chromosome, is shown in that AZFc district lacks.
Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.
SEQUENCE LISTING
<110> First Hospital of Ningbo City
The method that the micro-deleted rapid gene of <120> Y chromosome detects
<130> NB001
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> ZFY-F
<400> 1
accrctgtac tgactgtgat tacac 25
<210> 2
<211> 25
<212> DNA
<213> ZFY-R
<400> 2
gcacytcttt ggtatcygag aaagt 25
<210> 3
<211> 20
<212> DNA
<213> SRY-F
<400> 3
gaatattccc gctctccgga 20
<210> 4
<211> 20
<212> DNA
<213> SRY-R
<400> 4
gctggtgctc cattcttgag 20
<210> 5
<211> 20
<212> DNA
<213> SY254-F
<400> 5
gggtgttacc agaaggcaaa 20
<210> 6
<211> 22
<212> DNA
<213> SY254-R
<400> 6
gaaccgtatc taccaaagca gc 22
<210> 7
<211> 21
<212> DNA
<213> SY86-F
<400> 7
gtgacacaca gactatgctt c 21
<210> 8
<211> 20
<212> DNA
<213> SY86-R
<400> 8
acacacagag ggacaaccct 20
<210> 9
<211> 20
<212> DNA
<213> SY127-F
<400> 9
ggctcacaaa cgaaaagaaa 20
<210> 10
<211> 20
<212> DNA
<213> SY127-R
<400> 10
ctgcaggcag taataaggga 20
<210> 11
<211> 25
<212> DNA
<213> ZFY-F
<400> 11
accrctgtac tgactgtgat tacac 25
<210> 12
<211> 25
<212> DNA
<213> ZFY-R
<400> 12
gcacytcttt ggtatcygag aaagt 25
<210> 13
<211> 20
<212> DNA
<213> SRY-F
<400> 13
gaatattccc gctctccgga 20
<210> 14
<211> 20
<212> DNA
<213> SRY-R
<400> 14
gctggtgctc cattcttgag 20
<210> 15
<211> 20
<212> DNA
<213> SY84-F
<400> 15
agaagggtct gaaagcaggt 20
<210> 16
<211> 20
<212> DNA
<213> SY84-R
<400> 16
gcctactacc tggaggcttc 20
<210> 17
<211> 20
<212> DNA
<213> SY134-F
<400> 17
gtctgcctca ccataaaacg 20
<210> 18
<211> 20
<212> DNA
<213> SY134-R
<400> 18
accactgcca aaactttcaa 20
<210> 19
<211> 20
<212> DNA
<213> SY255-F
<400> 19
gttacaggat tcggcgtgat 20
<210> 20
<211> 18
<212> DNA
<213> SY255-R
<400> 20
ctcgtcatgt gcagccac 18
Claims (8)
1. the Amplification thing of the micro-deleted rapid gene detection of Y chromosome, it is characterized in that: this Amplification thing comprises AB two groups, often group comprises each pair of primers in AZFa, AZFb, AZFc region and two couples of internal reference primer ZFY and SRY, and the combination of primers often in group is as follows:
A group:
Internal reference ZFY primer pair, it has the nucleotide sequence as shown in SEQ ID No.1 and SEQ ID No.2;
Internal reference SRY primer pair, it has the nucleotide sequence as shown in SEQ ID No.3 and SEQ ID No.4;
SY254 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.5 and SEQ ID No.6;
SY86 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.7 and SEQ ID No.8;
SY127 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.9 and SEQ ID No.10;
B group:
Internal reference ZFY primer pair, it has the nucleotide sequence as shown in SEQ ID No.11 and SEQ ID No.12;
Internal reference SRY primer pair, it has the nucleotide sequence as shown in SEQ ID No.13 and SEQ ID No.14;
SY84 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.15 and SEQ ID No.16;
SY134 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.17 and SEQ ID No.18;
SY255 site primer pair, it has the nucleotide sequence as shown in SEQ ID No.19 and SEQ ID No.20.
2. the micro-deleted detection kit of rapid detection male Y chromosome, is characterized in that: described test kit comprises: Amplification thing described in claim 1, PCR reaction buffer, dNTPs, archaeal dna polymerase.
3. test kit according to claim 2, is characterized in that: PCR reaction buffer comprises 300mM Ttricine, 25mM (NH
4)
2sO
4, 17.5 mM MgCl
2, 30% glycerine.
4. test kit according to claim 2, is characterized in that: the archaeal dna polymerase of employing is saltant type Tag enzyme.
5. test kit according to claim 2, is characterized in that: dNTPs concentration is 2.5mM.
6. utilize the method that described in claim 2, test kit rapid detection male Y chromosome is micro-deleted, it is characterized in that described method mainly comprises the following steps:
(1) peripheral blood collection: extract peripheral blood 1ml mixing with anticoagulant heparin pipe for subsequent use;
(2) direct peripheral blood carries out multiplexed PCR amplification as substrate, carries out two groups of multi-PRC reactions, often overlaps reaction system and carries out multi-PRC reaction by Amplification thing according to claim 1;
(3) agarose gel electrophoresis detects PCR primer: get 10 μ l PCR primer, and after TAE dyeing, through 1.5% agarose gel electrophoresis, electrophoretic voltage 120V, electrophoresis time 90 minutes, by gel imaging instrument observation analysis.
7. method according to claim 6, is characterized in that: the PCR reaction system of 25 μ l comprises: PCR reaction buffer: 300mM Ttricine, 25mM (NH
4)
2sO
4, 17.5 mM MgCl
2, 30% glycerine, 5 μ l; Whole blood 2.5 μ l; 2 μ l 2.5mM dNTPs; 1 μ l 100 units/μ l saltant type Tag enzyme; 1.25-2.5 μM mixed assemblage primer 2 .5 μ l; 12 μ l H
2o.
8. method according to claim 6, is characterized in that: PCR cycling condition is: 95 DEG C of initial denaturation 5 min, 95 DEG C of sex change 30 s, 58 DEG C of annealing 90 s, 72 DEG C extend 60 s, and after 35 reaction cycle, 72 DEG C extend 15 min, preserve sample for 4 DEG C and use in order to next step experiment.
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| CN201510023696.4A CN104673893A (en) | 2015-01-16 | 2015-01-16 | Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510023696.4A CN104673893A (en) | 2015-01-16 | 2015-01-16 | Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof |
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ID=53309479
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|---|---|---|---|
| CN201510023696.4A Pending CN104673893A (en) | 2015-01-16 | 2015-01-16 | Amplified composition for fast gene detection of microdeletion of Y chromosomes, kit containing same and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957616A (en) * | 2019-04-16 | 2019-07-02 | 北京和合医学诊断技术股份有限公司 | The Amplification object and kit of the micro-deleted detection of Y chromosome |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101092648A (en) * | 2007-05-10 | 2007-12-26 | 亚能生物技术（深圳）有限公司 | Kit for testing tiny gene loss of male Y chromosome, and testing method |
| CN101575647A (en) * | 2009-06-19 | 2009-11-11 | 成都军区昆明总医院 | Detection method for Y chromosome micro-deleted gene |
| CN101701248A (en) * | 2009-03-23 | 2010-05-05 | 深圳博睿祥晖生物技术有限公司 | SYBR GREEN I real-time fluorescence quantitative PCR detection kit of Y chromosome microdeletion |
| CN102703578A (en) * | 2011-07-06 | 2012-10-03 | 郭奇伟 | Multiple real-time fluorescent PCR detection kit for Y chromosome microdeletion |
| CN102796819A (en) * | 2012-07-26 | 2012-11-28 | 南昌艾迪康临床检验所有限公司 | Kit for detecting microdeletion of Y chromosome |
| CN102876776A (en) * | 2012-09-04 | 2013-01-16 | 骆子义 | Real-time fluorescence quantification PCR (Polymerase Chain Reaction) kit and method for detecting Y-chromosome micro-deletion |
| CN102912028A (en) * | 2012-11-06 | 2013-02-06 | 北京阅微基因技术有限公司 | Amplification composite for detecting microdeletion of Y-chromosome and detection kit |
| CN103571957A (en) * | 2013-11-04 | 2014-02-12 | 北京海思特临床检验所有限公司 | Primer and kit for detecting microdeletion of chromosome Y and use method of primer |
| CN104152573A (en) * | 2014-08-26 | 2014-11-19 | 亚能生物技术（深圳）有限公司 | Kit for detecting human Y-chromosome microdeletion |
| CN104232779A (en) * | 2014-09-23 | 2014-12-24 | 亚能生物技术（深圳）有限公司 | Kit for detecting microdeletion of human Y chromosome |
-
2015
- 2015-01-16 CN CN201510023696.4A patent/CN104673893A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101092648A (en) * | 2007-05-10 | 2007-12-26 | 亚能生物技术（深圳）有限公司 | Kit for testing tiny gene loss of male Y chromosome, and testing method |
| CN101701248A (en) * | 2009-03-23 | 2010-05-05 | 深圳博睿祥晖生物技术有限公司 | SYBR GREEN I real-time fluorescence quantitative PCR detection kit of Y chromosome microdeletion |
| CN101575647A (en) * | 2009-06-19 | 2009-11-11 | 成都军区昆明总医院 | Detection method for Y chromosome micro-deleted gene |
| CN102703578A (en) * | 2011-07-06 | 2012-10-03 | 郭奇伟 | Multiple real-time fluorescent PCR detection kit for Y chromosome microdeletion |
| CN102796819A (en) * | 2012-07-26 | 2012-11-28 | 南昌艾迪康临床检验所有限公司 | Kit for detecting microdeletion of Y chromosome |
| CN102876776A (en) * | 2012-09-04 | 2013-01-16 | 骆子义 | Real-time fluorescence quantification PCR (Polymerase Chain Reaction) kit and method for detecting Y-chromosome micro-deletion |
| CN102912028A (en) * | 2012-11-06 | 2013-02-06 | 北京阅微基因技术有限公司 | Amplification composite for detecting microdeletion of Y-chromosome and detection kit |
| CN103571957A (en) * | 2013-11-04 | 2014-02-12 | 北京海思特临床检验所有限公司 | Primer and kit for detecting microdeletion of chromosome Y and use method of primer |
| CN104152573A (en) * | 2014-08-26 | 2014-11-19 | 亚能生物技术（深圳）有限公司 | Kit for detecting human Y-chromosome microdeletion |
| CN104232779A (en) * | 2014-09-23 | 2014-12-24 | 亚能生物技术（深圳）有限公司 | Kit for detecting microdeletion of human Y chromosome |
Non-Patent Citations (1)
| Title |
|---|
| 宋宁宏等: "严重少精子和无精子的不育患者Y染色体微缺失的检测", 《中华医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957616A (en) * | 2019-04-16 | 2019-07-02 | 北京和合医学诊断技术股份有限公司 | The Amplification object and kit of the micro-deleted detection of Y chromosome |
| CN109957616B (en) * | 2019-04-16 | 2022-07-29 | 北京和合医学诊断技术股份有限公司 | Amplification composition and kit for detecting microdeletion of Y chromosome |
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