CN103757091A - Rapid gene detection kit and rapid gene detection method for sudden cardiac death - Google Patents
Rapid gene detection kit and rapid gene detection method for sudden cardiac death Download PDFInfo
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Abstract
The present invention discloses a rapid gene detection kit for sudden cardiac death. The kit comprises 7 SNP polymorphism detections corresponding to genes such as KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C and CACNB2B. The present invention further discloses a rapid gene detection method for sudden cardiac death. According to the present invention, semi-nested AS-PCR amplification and agarose gel electrophoresis detection are performed, and genotyping is performed according to the DNA electrophoresis bands to screen the individual with the potential sudden cardiac death risk so as to achieve the purpose of sudden cardiac death prevention; and the method has characteristics of simple operation, short detection time, low cost, high sensitivity, strong specificity, good social benefits and broad application prospects.
Description
Technical field:
The present invention relates to biological gene detection technique field, be specially a kind of sudden cardiac death rapid gene detection kit and detection method.
Background technology:
Sudden cardiac death (sudden cardiac death, SCD) refers to because heart generation electrocardio is disorderly or in heart failure and makes heart lose the sudden death that effective contraction occurs.In recent years, SCD case takes place frequently, and its quantity is the situation rising year by year.At present, to the conventional sense means of SCD, be mainly that family history, personal history are carried out to examination clinically, carry out physique and Electrocardioscopy simultaneously, if there is positive findings, further look into ultrasonic cardiogram, exercise stress test and 24 hr Ambulatory EKG Monitoring etc.But these detection means specific aims are not strong, and recall rate is low, have larger limitation.
Both at home and abroad the research of sudden cardiac death being shown, there is the phenomenon of some transgenation in the dead in genomic level, mostly show as SNP or base deletion polymorphism.SCD has that onset is anxious, progress is fast, case fatality rate high, and therefore, the generation of evading SCD by early gene examination, risk assessment just seems particularly important.
At present, sudden cardiac death and gene pleiomorphism cognation achievement in research make SCD gene screening become possibility.Research shows, KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, 7 genes and sudden cardiac death significant correlation.In 7 genes, KCNE1, KCNH2, KCNJ11 belong to potassium-channel genes involved, and SCN5A belongs to sodium-ion channel genes involved, and CACNA1C, CACNB2B belong to calcium channel genes involved.These ionic channels play an important role at myocardial action potential different times, and its transgenation can affect the structure and function of ionic channel conventionally.NUP155 is the gene of a coding nuclear Pore Complex component, its major function be control genetic material mRNA by nucleus to cytoplasmic transhipment, one in the situation monitoring gene of upstream relatively, can regulate and control other a lot of genes and protein expression, thereby caused atrial fibrillation generation, conventionally threatened the health of Young crowd.Some trickle changes of NUP155 gene, also likely increase the danger that common sporadic atrial fibrillation occurs.
In China Han sudden cardiac death crowd, extensively exist KCNE1, KCNH2, KCNJ11 single nucleotide polymorphism to change, thereby cause the sudden death of ventricular tachycardia, therefore, the sudden death that potassium-channel genes involved causes belongs to high-risk genic mutation type.The demonstration of clinical study result, the sudden death that sodium-ion channel genes involved SCN5A sudden change causes accounts for 1-5 ‰ in sudden cardiac death crowd; The sudden cardiac death that calcium channel genes involved CACNA1C, CACNB2B sudden change causes accounts for 0.1-0.3 ‰, belongs to low degree of hazard saltant type; The gene NUP155 gene of coding nuclear Pore Complex component is that in Chinese han population, find and peculiar gene sudden cardiac death significant correlation, thus its sudden change of examination for Chinese population, sudden cardiac death has special meaning.
Summary of the invention:
The object of the invention is take above-mentioned prior art as basis, provide that a kind of operation is simple and easy, testing cost is low, to sudden cardiac death, can play the sudden cardiac death rapid gene detection kit of prophylactic effect.
Another object of the present invention is to provide a kind of sudden cardiac death high-speed gene test method.
In order to realize the object of the invention, the technical scheme that the present invention takes is: sudden cardiac death rapid gene detection kit, and the minimum primer mixture that detects KCNE1, KCNH2, KCNJ11 gene polymorphic site that comprises,
Wherein, the primer mixture in detection KCNE1 gene polymorphic site is:
SEQ NO.1:5 '-GTGCCTGGGAAGTTTGAGC-3 ' (sequence 1);
SEQ NO.2:5 '-GCAGGTCCCCCCGCAGAA-3 ' (sequence 2);
SEQ NO.3:5 '-GCAGGTCCCCCCGCAGAG-3 ' (sequence 3);
SEQ NO.4:5 '-CCGTTCTTTCCCAGTCTCAT-3 ' (sequence 4);
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; When detecting, the consumption of the above-mentioned primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul.
The primer mixture that detects KCNH2 gene polymorphic site is:
SEQ NO.1:5 '-GCCGTGCTGTTCTTGCTC-3 ' (sequence 5);
SEQ NO.2:5 '-AGGACAAGTATGTGACGGCTC-3 ' (sequence 6);
SEQ NO.3:5 '-AGGACAAGTATGTGACGGCTT-3 ' (sequence 7);
SEQ NO.4:5 '-GATTGGGGATCTGGTGGAA-3 ' (sequence 8).
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1, when detecting, the consumption of the above-mentioned primer primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul;
The primer mixture that detects KCNJ11 gene polymorphic site is:
SEQ NO.1:5 '-CTCATCGTGCAGAACATCGTG-3 ' (sequence 9);
SEQ NO.2:5 '-CGCAAGAGCATGATCATCCA-3 ' (sequence 10);
SEQ NO.3:5 '-CGCAAGAGCATGATCATCCG-3 ' (sequence 11);
SEQ NO.4:5 '-TGGCCGGGCTACATACCA-3 ' sequence 12);
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; When detecting, the consumption of the above-mentioned primer primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul;
Also comprise the primer mixture that detects SCN5A gene polymorphic site, wherein, the primer mixture that detects SCN5A gene polymorphic site is:
SEQ NO.1:5 '-ACAGTGATGCTGGCTGGAAG-3 ' (sequence 13);
SEQ NO.2:5 '-GAAGTACTTCTTCTCCCCGTC-3 ' (sequence 14);
SEQ NO.3:5 '-GAAGTACTTCTTCTCCCCGTT-3 ' (sequence 15);
SEQ NO.4:5 '-ATGTTGATGAGGCTTATCTGGTT-3 ' (sequence 16);
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.2-0.5:1:1; When detecting, the consumption of the above-mentioned SEQ NO.1 primer that concentration is 10uM is 0.1ul-0.3ul, the above-mentioned SEQ NO.2 that concentration is 10uM, and SEQ NO.3 and SEQ NO.4 primer consumption are 0.3ul-0.7ul.
Also comprise the primer mixture that detects CACNA1C, CACNB2B gene polymorphic site, wherein, the primer mixture that detects CACNA1C gene polymorphic site is:
SEQ NO.1:5 '-GCCTGCAATAGCTTGAAATAAGG-3 ' (sequence 17);
SEQ NO.2:5 '-GGTGGGGATGTGCTCAGGTA-3 ' (sequence 18);
SEQ NO.3:5 '-GTGGGGATGTGCTCAGGTG-3 ' (sequence 19);
SEQ NO.4:5 '-AGAAGGGAAAGAAGGGAATGAG-3 ' (sequence 20);
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:0.8-1.2:1; When detecting, the consumption of the above-mentioned primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul;
The primer mixture that detects CACNB2B gene polymorphic site is:
SEQ NO.1:5 '-GCACTTGCTCTGGGACAT-3 ' (sequence 21);
SEQ NO.2:5 '-CCCAGCACCGCTCTTCCGC-3 ' (sequence 22);
SEQ NO.3:5 '-TCCCAGCACCGCTCTTCCGT-3 ' (sequence 23);
SEQ NO.4:5 '-TGATTCTGGCTTGTTGGATA-3 ' (sequence 24);
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.125:1:1; When detecting, the consumption of the above-mentioned SEQ NO.1 primer that concentration is 10uM is 0.05ul-0.2ul, the above-mentioned primer SEQ NO.2 that concentration is 10uM, and SEQ NO.3 and SEQ NO.4 consumption are 0.6ul-1ul.
Also comprise and detect NUP155 gene polymorphic site, wherein, the primer mixture that detects NUP155 gene polymorphic site is:
SEQ NO.1:5 '-GGAGAATCGCTTGAACCC-3 ' (sequence 25);
SEQ NO.2:5 '-CTGACGCTGGTTCATGTCAA-3 ' (sequence 26);
SEQ NO.3:5 '-CTGACGCTGGTTCATGTCAG-3 ' (sequence 27);
SEQ NO.4:5 '-TATTGCGTGGTGCTAAGGTT-3 ' (sequence 28);
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.4:1:1; When detecting, the consumption of the above-mentioned SEQ NO.1 primer that concentration is 10uM is 0.1ul-0.3ul, the above-mentioned primer SEQ NO.2 that concentration is 10uM, and the consumption of SEQ NO.3 and SEQ NO.4 is 0.3ul-0.7ul.
Described sudden cardiac death rapid gene detection kit also comprises amplifing reagent, and described amplifing reagent comprises PCR damping fluid, dNTPs mixture, warm start Taq enzyme and ultrapure water;
Preferably, described amplifing reagent comprises 10 times of PCR damping fluids (wherein, Tris-HCl pH8.3100mM; KCl 500Mm; MgCl
215mM), the dNTPs mixture that each concentration of component is 2.5mM, warm start Taq enzyme (Taq HS) consumption of 5U/ μ l is 0.1-0.125 μ l.
Wherein, described PCR damping fluid (wherein, Tris-HCl pH8.3 100mM; KCl 500Mm; MgCl2 15mM), each concentration of component of described dNTPs mixture is 2.5mM, and warm start Taq enzyme (Taq HS) is 5U/ μ l.
Described test kit also comprises the gene type reagent after amplification, and the gene type after described amplification comprises agarose, TAE damping fluid, DNA molecular amount standard (DNA Marker), DNA non-toxic dye and DNA sample-loading buffer with reagent.Described DNA non-toxic dye can be Biotium Gelred non-toxic dye.
Preferably, amplifing reagent is comprised of the component with lower volume:
In above-mentioned KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, the corresponding primer of CACNB2B, described internal reference upstream primer is SEQ NO.1, and polymorphism primer is SEQ NO.2 and SEQ NO.3, and downstream primer is SEQ NO.4.The concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM, and the concentration of described downstream primer is 10uM.For described KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, the consumption of described internal reference upstream primer, polymorphism primer and downstream primer is by the above.
Sudden cardiac death high-speed gene test method, its step comprises:
(1), the genomic DNA extraction of sample;
(2), the detection analysis of amplification and amplified production;
Wherein, pcr amplification reagent is comprised of the component with lower volume:
In above-mentioned KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, the corresponding primer of CACNB2B, described internal reference upstream primer is SEQ NO.1, and polymorphism primer is SEQ NO.2 and SEQ NO.3, and downstream primer is SEQ NO.4.The concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM, and the concentration of described downstream primer is 10uM.For described KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, the consumption of described internal reference upstream primer, polymorphism primer and downstream primer is by the above.
Wherein, described PCR damping fluid (wherein, Tris-HCl pH8.3 100mM; KCl 500Mm; MgCl
215mM), each concentration of component of described dNTPs mixture is 2.5mM, and warm start Taq enzyme (Taq HS) is 5U/ μ l;
Pcr amplification program is as follows:
(3), do agarose gel electrophoresis analysis.
Described amplification adopts AS-PCR(allele-specific PCR) carry out, then adopt agarose gel electrophoresis and gel imaging system to carry out the detection of polymorphism somatotype.
Wherein, the human gene group DNA who detects is for using Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to process to source sample the DNA obtaining; Described source sample is filter paper blood cake/buccal swab sample, FTA card blood cake or the blood that derives from the mankind.
Wherein, primer SEQ NO.1 is internal reference upstream primer; 3 of SEQ NO.2 and SEQ NO.3 ' end second base is respectively base mismatch, for detecting upstream primer; SEQ NO.4 is internal reference and detects shared downstream primer.
By AS-PCR and agarose gel electrophoresis specific detection sudden cardiac death gene; Primer for gene test is: SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, the amplification of described gene adopts polymerase chain reaction to realize, and adopts agarose gel electrophoresis to detect.
The present invention chooses KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B, 7 with the polymorphic site of the gene of sudden cardiac death significant correlation as examination target spot, application AS-PCR technology realizes rapid detection, the potential sudden death risk individuality of SCD risk genes polymorphic site is carried in examination, thereby reaches the object of prevention SCD generation.
The present invention detects the SNP(single nucleotide polymorphism of KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, a CACNB2B7 gene simultaneously) polymorphic site.The detection of each SNP needs 4 PCR primers (a pair of upstream and downstream primer, two polymorphic primers), need to design altogether 28 primers.The detection of each SNP need to be done two pipe pcr amplifications, and 7 SNP add the cloudy control of a pipe, are altogether 15 pipe PCR.Because 15 pipe pcr amplification reactions synchronously carry out on same PCR instrument, the high efficiency detecting in order to realize sudden cardiac death, specificity, require the Tm value (annealing temperature) of 36 PCR primers close, in the design of PCR primer, need to do a large amount of DAN sequence alignment software analytical works, the optimization of Tm value need to be determined by a large amount of experimental results the reasonableness of design for this reason.Based on above reason, the detection of each gene SNP polymorphism of this test kit is not only relatively independent but also connect each other, indivisible.
Beneficial effect of the present invention is as follows: by half-nest type AS-PCR amplification and agarose gel electrophoresis, detect, according to DNA electrophoretic band, carry out gene type, filter out the individuality with potential sudden cardiac death risk, reach the object of prevention sudden cardiac death; This method is easy and simple to handle, and detection time is short, cost is low, highly sensitive, and high specificity has good social benefit, has a extensive future.
Accompanying drawing explanation
Fig. 1 is sudden cardiac death rapid gene detection kit gene type electrophorogram 1 of the present invention;
Fig. 2 is that sudden cardiac death rapid gene detection kit gene type electrophorogram 2(of the present invention accepts Fig. 1);
Fig. 3 is that sudden cardiac death rapid gene detection kit of the present invention is to 7,22,13, No. 35 sequencer maps corresponding to sample in each typing gene polymorphisms electrophorogram.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail to explanation.
Sudden cardiac death rapid gene detection kit, comprises the gene type reagent after amplifing reagent and amplification;
Described amplifing reagent comprises PCR damping fluid, dNTPs mixture, warm start Taq enzyme, primer mixture and ultrapure water; Gene type reagent after described amplification: reagent agarose, TAE damping fluid, DNA molecular amount standard, DNA non-toxic dye and DNA sample-loading buffer.Described DNA sample-loading buffer (DNA loading buffer, 6X), suitably six times of conventional concentrated DNA sample-loading buffers of improvement of a kind of process, take tetrabromophenol sulfonphthalein as indicator, after being diluted to 1X, proportion is still larger, during loading, very easily sink, and color is high-visible, this DNA sample-loading buffer can buy on market.
Described amplifing reagent comprises that amplifing reagent comprises 10 times of PCR damping fluids (wherein, Tris-HCl pH8.3 100mM; KCl 500Mm; MgCl
215mM), the dNTPs mixture that each concentration of component is 2.5mM, warm start Taq enzyme (Taq HS) consumption of 5U/ μ l is 0.1-0.125 μ l.Preferably, warm start Taq enzyme (Taq HS) consumption of 5U/ μ l is 0.125 μ l.Wherein, dNTP mixture is triphosphoric acid guanine deoxyribonucleoside acid dGTP, triphosphoric acid adenyl-deoxyribonucleotide dATP, and triphosphoric acid thymidylic acid dTTP, in triphosphoric acid deoxycytidylic acid dCTP tetra-, Nucleotide forms.
Sudden cardiac death rapid gene detection kit, also comprises KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, the corresponding primer mixture of CACNB2B.
Wherein, the primer mixture in detection KCNE1 gene polymorphic site is:
SEQ?NO.1：5’-GTGCCTGGGAAGTTTGAGC-3’；
SEQ?NO.2：5’-GCAGGTCCCCCCGCAGAA-3’；
SEQ?NO.3：5’-GCAGGTCCCCCCGCAGAG-3’；
SEQ?NO.4：5’-CCGTTCTTTCCCAGTCTCAT-3’；
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; Or when detecting, the consumption of the above-mentioned primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul.
The primer mixture that detects KCNH2 gene polymorphic site is:
SEQ?NO.1：5’-GCCGTGCTGTTCTTGCTC-3’；
SEQ?NO.2：5’-AGGACAAGTATGTGACGGCTC-3’；
SEQ?NO.3：5’-AGGACAAGTATGTGACGGCTT-3’；
SEQ?NO.4：5’-GATTGGGGATCTGGTGGAA-3’；
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1, or when detecting, the consumption of the above-mentioned primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul.
The primer mixture that detects SCN5A gene polymorphic site is:
SEQ?NO.1：5’-ACAGTGATGCTGGCTGGAAG-3’；
SEQ?NO.2：5’-GAAGTACTTCTTCTCCCCGTC-3’；
SEQ?NO.3：5’-GAAGTACTTCTTCTCCCCGTT-3’；
SEQ?NO.4：5’-ATGTTGATGAGGCTTATCTGGTT-3’。
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.2-0.5:1:1; Or when detecting, the consumption of the above-mentioned SEQ NO.1 primer that concentration is 10uM is 0.1ul-0.3ul, the above-mentioned primer SEQ NO.2 that concentration is 10uM, SEQ NO.3 and SEQ NO.4 consumption are 0.3ul-0.7ul.
The primer mixture that detects KCNJ11 gene polymorphic site is:
SEQ?NO.1：5’-CTCATCGTGCAGAACATCGTG-3’；
SEQ?NO.2：5’-CGCAAGAGCATGATCATCCA-3’；
SEQ?NO.3：5’-CGCAAGAGCATGATCATCCG-3’；
SEQ?NO.4：5’-TGGCCGGGCTACATACCA-3’。
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; Or when detecting, the consumption of the above-mentioned primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul.
The primer mixture that detects CACNA1C gene polymorphic site is:
SEQ?NO.1：5’-GCCTGCAATAGCTTGAAATAAGG-3’；
SEQ?NO.2：5’-GGTGGGGATGTGCTCAGGTA-3’；
SEQ?NO.3：5’-GTGGGGATGTGCTCAGGTG-3’；
SEQ?NO.4：5’-AGAAGGGAAAGAAGGGAATGAG-3’；
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:0.8-1.2:1; Or when detecting, the consumption of the above-mentioned primer that concentration is 10uM (SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4) is 0.3ul-0.7ul;
The primer mixture that detects CACNB2B gene polymorphic site is:
SEQ?NO.1：5’-GCACTTGCTCTGGGACAT-3’；
SEQ?NO.2：5’-CCCAGCACCGCTCTTCCGC-3’；
SEQ?NO.3：5’-TCCCAGCACCGCTCTTCCGT-3’；
SEQ?NO.4：5’-TGATTCTGGCTTGTTGGATA-3’；
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.125:1:1;
Or when detecting, the consumption of the above-mentioned SEQ NO.1 primer that concentration is 10uM is 0.05ul-0.2ul, the above-mentioned primer SEQ NO.2 that concentration is 10uM, SEQ NO.3 and SEQ NO.4 consumption are 0.6ul-1ul.
The primer mixture that detects NUP155 gene polymorphic site is:
SEQ?NO.1：5’-GGAGAATCGCTTGAACCC-3’；
SEQ?NO.2：5’-CTGACGCTGGTTCATGTCAA-3’；
SEQ?NO.3：5’-CTGACGCTGGTTCATGTCAG-3’；
SEQ?NO.4：5’-TATTGCGTGGTGCTAAGGTT-3’。
And mole ratio is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.4:1:1; Or when detecting, the consumption of the above-mentioned SEQ NO.1 primer that concentration is 10uM is 0.1ul-0.3ul, the above-mentioned primer SEQ NO.2 that concentration is 10uM, the consumption of SEQ NO.3 and SEQ NO.4 is 0.3ul-0.7ul.
Preferably, detect the primer mixture SEQ NO.1 in KCNE1 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul; Detect the primer mixture SEQ NO.1 in KCNH2 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul; Detect the primer mixture SEQ NO.1 in KCNJ11 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul; In the primer mixture in detection SCN5A gene polymorphic site, the consumption of SEQ NO.1 primer is 0.1ul, SEQ NO.2, and SEQ NO.3 and SEQ NO.4 primer consumption are respectively 0.3ul; Detect the primer mixture SEQ NO.1 in CACNA1C gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul; The consumption that detects the primer mixture SEQ NO.1 primer in CACNB2B gene polymorphic site is 0.05ul, SEQ NO.2, and SEQ NO.3 and SEQ NO.4 consumption are respectively 0.6ul; The consumption that detects the primer mixture SEQ NO.1 primer in NUP155 gene polymorphic site is 0.1ul, SEQ NO.2, and the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul.
Preferably, detect the primer mixture SEQ NO.1 in KCNE1 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.7ul; Detect the primer mixture SEQ NO.1 in KCNH2 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.7ul; Detect the primer mixture SEQ NO.1 in KCNJ11 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.7ul; In the primer mixture in detection SCN5A gene polymorphic site, the consumption of SEQ NO.1 primer is 0.3ul, SEQ NO.2, and SEQ NO.3 and SEQ NO.4 primer consumption are respectively 0.7ul; Detect the primer mixture SEQ NO.1 in CACNA1C gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.7ul; The consumption that detects the primer mixture SEQ NO.1 primer in CACNB2B gene polymorphic site is 0.2ul, SEQ NO.2, and SEQ NO.3 and SEQ NO.4 consumption are respectively 1ul; The consumption that detects the primer mixture SEQ NO.1 primer in NUP155 gene polymorphic site is 0.3ul, SEQ NO.2, and the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.7ul.
The detection in the corresponding SNP of each gene site needs 4 primers to complete, owing to needing to increase internal reference band and polymorphism object band in a pcr amplification system simultaneously, therefore, can produce PCR competitive reaction, in order to improve the resolving power of internal reference band and polymorphic bands, need to by experiment the ratio of the mole number/volume of the mole number/volume of internal reference primer and polymorphic primer be adjusted to optimum proportion, resolving power while detecting to improve.
Amplifing reagent is comprised of the component with lower volume:
Described internal reference upstream primer is SEQ NO.1, and polymorphism primer is SEQ NO.2 and SEQ NO.3, and downstream primer is SEQ NO.4, and the concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM, and the concentration of described downstream primer is 10uM.
Pcr amplification program is as follows:
(3), do agarose gel electrophoresis analysis.
Amplifing reagent is comprised of the component with lower volume:
Amplifing reagent is comprised of the component with lower volume:
In order to verify above-mentioned information, can the detection analysis on ABI3130 gene genetic analyser by amplified production, to verify the above-mentioned detected result of the present invention.
By mark in deionized formamide and molecular weight, form loading mixture, 10ul loading mixture is mixed with 1ul amplified production or alleles analysis standard substance, avoid producing bubble, 95 ℃ of sex change 5min, do electrophoresis, with ABI3130 gene genetic analyser, detect analysis verification analytical results.
Described amplification adopts AS-PCR(allele-specific PCR) carry out, adopt agarose gel electrophoresis to carry out the detection of polymorphism somatotype.
The human gene group DNA that wherein detected is for using Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to process to source sample the DNA obtaining; Described source sample is filter paper blood cake/buccal swab sample, FTA card blood cake or the blood that derives from the mankind.
By AS-PCR and agarose gel electrophoresis specific detection sudden cardiac death gene, the primer of KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, CACNB2B gene is SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, the amplification of described gene adopts polymerase chain reaction to realize, and adopts agarose gel electrophoresis to detect.
Test kit of the present invention detects 50 DNA samples that sudden death is individual.
1, sample to be tested is 50 parts, and the technological method that all uses the above " DNA extraction-pcr amplification-order-checking " is to the gene detection of checking order.Wherein No. 7, No. 13, No. 22, No. 35 sample detects positive findings.
2, the extracting genome DNA of sample
Chelex extraction method: cut 1～3mm blood cake and be placed in 1.5ml centrifuge tube, add sdH
2o1ml, vibrates centrifugal, abandons supernatant, twice of repeating step, abandon supernatant, draw 200ul fast add in centrifuge tube after 5%Chelex-100 vibration is suspended with the rifle head of cutting head, the vibration several seconds, after 56 ℃ of water bath heat preservation 30min, the vibration several seconds, 95 ℃ of boiling water bath 10min, slightly vibrate the several seconds.The centrifugal 5min of 2000rpm, the DNA of supernatant for extracting.
3, the detection analysis of amplification and amplified production
3.1PCR amplification system:
Described internal reference upstream primer is SEQ NO.1, and polymorphism primer is SEQ NO.2 and SEQ NO.3, and downstream primer is SEQ NO.4, and the concentration of described internal reference upstream primer is 10uM, and the concentration of described polymorphism primer is 10uM, and the concentration of described downstream primer is 10uM.
3.2PCR amplification program:
Described amplification adopts AS-PCR(allele-specific PCR) carry out, adopt agarose gel electrophoresis and gel imaging system to carry out the detection of polymorphism somatotype.
4, the detection analysis verification of amplified production on ABI3130 gene genetic analyser
By mark in deionized formamide and molecular weight, form loading mixture (mark * sample introduction book+9.8ul deionized formamide * sample introduction number in 0.2ul molecular weight).10ul loading mixture is mixed with 1ul amplified production or alleles analysis standard substance, avoid producing bubble.95 ℃ of sex change 5min, and electrophoresis as early as possible, detect and analyze with ABI3130 gene genetic analyser.
5, conclusion
Above-described embodiment somatotype is complete clear, and result as depicted in figs. 1 and 2.Wherein, use M.DL1000DNA molecular weight standard; 1-6 swimming lane is the pcr amplification product of KCNE1 gene; 7-10 swimming lane is the pcr amplification product of KCNH2 gene; 11-16 swimming lane is the pcr amplification product of SCN5A gene; 17-20 swimming lane is the pcr amplification product of KCNJ11 gene; 21-24 swimming lane is the pcr amplification product of CACNA1C gene; 25-28 swimming lane is the pcr amplification product of CACNB2B gene; 29-32 swimming lane is the pcr amplification product of NUP155 gene.7,8,11,12,25,26 swimming lane gene types are CC type; 9,10,13,14,27,28 swimming lane gene types are CT type; 15,16 swimming lane gene types are TT type; 1,2 swimming lane gene types are AA type; 3,4,19,20,23,24,31,31 swimming lane gene types are AG type; 5,6,17,18,21,22,29,30 swimming lane gene types are GG type.
In sudden cardiac death gene type electrophorogram, the reference clip size of KCNE1 gene is 894bp, and object stripe size is 754bp; The reference clip size of KCNH2 gene is 760bp, and object stripe size is 579bp; The reference clip size of KCNJ11 gene is 795bp, and object stripe size is 624bp; The reference clip size of SCN5A gene is 764bp, and object stripe size is 698bp; The reference clip size of CACNA1C gene is 851bp, and object stripe size is 203bp; The reference clip size of CACNB2B gene is 914bp, and object stripe size is 750bp; The reference clip size of NUP155 gene is 610bp, and object stripe size is 321bp.
As Fig. 1, shown in 2, normal people's genotype is: KCNE1 Gene A A type, KCNH2 gene C C type, KCNJ11 gene GG type, SCN5A gene C C type, CACNA1C gene GG type, CACNB2B gene C C type, NUP155 gene GG type.Other genotype in figure are the positive sample electrophoresis result that this laboratory uses site-directed mutagenesis technique clone.
As shown in Figure 3, in No. 7 sample sudden cardiac death gene type electrophorograms, A swimming lane and left and right, G swimming lane 700bp position occur that object expands once band; size is coincide with reason object band theoretical value 698bp; inspection is for AG heterozygosis, consistent with sequencing result, shows that this individuality exists higher sudden death risk; In No. 22 sample sudden cardiac death gene type electrophorograms, left and right, G swimming lane 700bp position occurs that object expands once band, and size is coincide with reason object band theoretical value 698bp; A swimming lane occurs without object band; examine as GG isozygotys, consistent with sequencing result, show that this individuality exists very high sudden death risk; In No. 13 sample sudden cardiac death gene type electrophorograms; C swimming lane and below, T swimming lane 700bp position occur that object expands once band, and size is coincide with reason object band theoretical value 579bp, examines the heterozygosis into CT; consistent with sequencing result, show that this individuality exists higher sudden death risk; In No. 35 sample sudden cardiac death gene type electrophorograms, A swimming lane and left and right, G swimming lane 300bp position occur that object expands once band, and size is coincide with reason object band theoretical value 321bp, examines the heterozygosis into AG, consistent with sequencing result, show that this individuality exists higher sudden death risk.
In above embodiment, Taq warm start polysaccharase used and other reagent and consumptive material are commercially available prod.
More than describe preferred embodiment of the present invention in detail, should be appreciated that the ordinary skill of this area just can design according to the present invention be made many modifications and variations without creative work.Therefore, all technician in the art according to the present invention design on prior art basis by logic analysis, reasoning or according to the available technical scheme of limited experiment, all should be among the determined protection domain by these claims.
Sequence table
The <110> Guangzhou Research Institute of Physical Culture
<120> sudden cardiac death rapid gene detection kit and detection method
<160>28
<210>1
<211>19
<212>DNA
<213> artificial sequence
<220>
<223>
<400>1
GTGCCTGGGA?AGTTTGAGC
<210>2
<211>18
<212>DNA
<213> artificial sequence
<220>
<223>
<400>2
GCAGGTCCCC?CCGCAGAA
<210>3
<211>18
<212>DNA
<213> artificial sequence
<220>
<223>
<400>3
GCAGGTCCCC?CCGCAGAG
<210>4
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>4
CCGTTCTTTC?CCAGTCTCAT
<210>5
<211>18
<212>DNA
<213> artificial sequence
<220>
<223>
<400>5
GCCGTGCTGT?TCTTGCTC
<210>6
<211>21
<212>DNA
<213> artificial sequence
<220>
<223>
<400>6
AGGACAAGTA?TGTGACGGCT?C
<210>7
<211>21
<212>DNA
<213> artificial sequence
<220>
<223>
<400>7
AGGACAAGTA?TGTGACGGCT?T
<210>8
<211>19
<212>DNA
<213> artificial sequence
<220>
<223>
<400>8
GATTGGGGAT?CTGGTGGAA
<210>9
<211>21
<212>DNA
<213> artificial sequence
<220>
<223>
<400>9
CTCATCGTGC?AGAACATCGT?G
<210>10
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>10
CGCAAGAGCA?TGATCATCCA
<210>11
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>11
CGCAAGAGCA?TGATCATCCG
<210>12
<211>18
<212>DNA
<213> artificial sequence
<220>
<223>
<400>12
TGGCCGGGCT?ACATACCA
<210>13
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>13
ACAGTGATGC?TGGCTGGAAG
<210>14
<211>21
<212>DNA
<213> artificial sequence
<220>
<223>
<400>14
GAAGTACTTC?TTCTCCCCGT?C
<210>15
<211>21
<212>DNA
<213> artificial sequence
<220>
<223>
<400>15
GAAGTACTTC?TTCTCCCCGT?T
<210>16
<211>23
<212>DNA
<213> artificial sequence
<220>
<223>
<400>16
ATGTTGATGA?GGCTTATCTG?GTT
<210>17
<211>23
<212>DNA
<213> artificial sequence
<220>
<223>
<400>17
GCCTGCAATA?GCTTGAAATA?AGG
<210>18
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>18
GGTGGGGATG?TGCTCAGGTA
<210>19
<211>19
<212>DNA
<213> artificial sequence
<220>
<223>
<400>19
GTGGGGATGT?GCTCAGGTG
<210>20
<211>22
<212>DNA
<213> artificial sequence
<220>
<223>
<400>20
AGAAGGGAAA?GAAGGGAATG?AG
<210>21
<211>18
<212>DNA
<213> artificial sequence
<220>
<223>
<400>21
GCACTTGCTC?TGGGACAT
<210>22
<211>19
<212>DNA
<213> artificial sequence
<220>
<223>
<400>22
CCCAGCACCG?CTCTTCCGC
<210>23
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>23
TCCCAGCACC?GCTCTTCCGT
<210>24
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>24
TGATTCTGGC?TTGTTGGATA
<210>25
<211>18
<212>DNA
<213> artificial sequence
<220>
<223>
<400>25
GGAGAATCGC?TTGAACCC
<210>26
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>26
CTGACGCTGG?TTCATGTCAA
<210>27
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>27
CTGACGCTGG?TTCATGTCAG
<210>28
<211>20
<212>DNA
<213> artificial sequence
<220>
<223>
<400>28
TATTGCGTGG?TGCTAAGGTT
Claims (10)
1. sudden cardiac death rapid gene detection kit, is characterized in that, detect KCNE1, KCNH2, KCNJ11 gene polymorphic site minimum comprising,
Wherein, the primer mixture in detection KCNE1 gene polymorphic site is:
SEQ?NO.1：5’-GTGCCTGGGAAGTTTGAGC-3’；
SEQ?NO.2：5’-GCAGGTCCCCCCGCAGAA-3’；
SEQ?NO.3：5’-GCAGGTCCCCCCGCAGAG-3’；
SEQ?NO.4：5’-CCGTTCTTTCCCAGTCTCAT-3’；
The primer mixture that detects KCNH2 gene polymorphic site is:
SEQ?NO.1：5’-GCCGTGCTGTTCTTGCTC-3’；
SEQ?NO.2：5’-AGGACAAGTATGTGACGGCTC-3’；
SEQ?NO.3：5’-AGGACAAGTATGTGACGGCTT-3’；
SEQ?NO.4：5’-GATTGGGGATCTGGTGGAA-3’；
The primer mixture that detects KCNJ11 gene polymorphic site is:
SEQ?NO.1：5’-CTCATCGTGCAGAACATCGTG-3’；
SEQ?NO.2：5’-CGCAAGAGCATGATCATCCA-3’；
SEQ?NO.3：5’-CGCAAGAGCATGATCATCCG-3’；
SEQ?NO.4：5’-TGGCCGGGCTACATACCA-3’。
2. sudden cardiac death rapid gene detection kit according to claim 1, is characterized in that, also comprises and detects SCN5A gene polymorphic site, and the primer mixture that wherein detects SCN5A gene polymorphic site is:
SEQ?NO.1：5’-ACAGTGATGCTGGCTGGAAG-3’；
SEQ?NO.2：5’-GAAGTACTTCTTCTCCCCGTC-3’；
SEQ?NO.3：5’-GAAGTACTTCTTCTCCCCGTT-3’；
SEQ?NO.4：5’-ATGTTGATGAGGCTTATCTGGTT-3’。
3. sudden cardiac death rapid gene detection kit according to claim 2, is characterized in that, also comprises and detects CACNA1C, CACNB2B gene polymorphic site, and wherein, the primer mixture that detects CACNA1C gene polymorphic site is:
SEQ?NO.1：5’-GCCTGCAATAGCTTGAAATAAGG-3’；
SEQ?NO.2：5’-GGTGGGGATGTGCTCAGGTA-3’；
SEQ?NO.3：5’-GTGGGGATGTGCTCAGGTG-3’；
SEQ?NO.4：5’-AGAAGGGAAAGAAGGGAATGAG-3’；
The primer mixture that detects CACNB2B gene polymorphic site is:
SEQ?NO.1：5’-GCACTTGCTCTGGGACAT-3’；
SEQ?NO.2：5’-CCCAGCACCGCTCTTCCGC-3’；
SEQ?NO.3：5’-TCCCAGCACCGCTCTTCCGT-3’；
SEQ?NO.4：5’-TGATTCTGGCTTGTTGGATA-3’。
4. sudden cardiac death rapid gene detection kit according to claim 3, is characterized in that, also comprises and detects NUP155 gene polymorphic site, and wherein, the primer mixture that detects NUP155 gene polymorphic site is:
SEQ?NO.1：5’-GGAGAATCGCTTGAACCC-3’；
SEQ?NO.2：5’-CTGACGCTGGTTCATGTCAA-3’；
SEQ?NO.3：5’-CTGACGCTGGTTCATGTCAG-3’；
SEQ?NO.4：5’-TATTGCGTGGTGCTAAGGTT-3’。
5. according to the sudden cardiac death rapid gene detection kit described in claim 1,2,3 or 4, it is characterized in that, the mole ratio that detects the primer mixture in KCNE1 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in KCNH2 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in KCNJ11 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in SCN5A gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.2-0.5:1:1; The mole ratio that detects the primer mixture in CACNA1C gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:0.8-1.2:1; The mole ratio that detects the primer mixture in CACNB2B gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.125:1:1; The mole ratio that detects the primer mixture in NUP155 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=0.4:1:1.
6. according to the sudden cardiac death rapid gene detection kit described in claim 1,2,3 or 4, it is characterized in that, the mole ratio that detects the primer mixture in KCNE1 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in KCNH2 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in KCNJ11 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in SCN5A gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in CACNA1C gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in CACNB2B gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1; The mole ratio that detects the primer mixture in NUP155 gene polymorphic site is SEQ NO.1:SEQ NO.2/SEQ NO.3:SEQ NO.4=1:1:1.
7. sudden cardiac death rapid gene detection kit according to claim 1, it is characterized in that, also comprise amplifing reagent and draw together the gene type reagent after amplification, described amplifing reagent comprises PCR damping fluid, dNTPs mixture, warm start Taq enzyme and ultrapure water; Gene type after described amplification comprises agarose, TAE damping fluid, DNA molecular amount standard, DNA non-toxic dye and DNA sample-loading buffer with reagent.
8. sudden cardiac death rapid gene detection kit according to claim 7, is characterized in that, described amplifing reagent comprises 10 times of PCR damping fluids: wherein, and Tris-HCl pH8.3100mM; KCl500Mm; MgCl
215mM, the dNTPs mixture that each concentration of component is 2.5mM, the warm start Taq enzyme dosage of 5U/ μ l is 0.1-0.125 μ l.
9. sudden cardiac death rapid gene detection kit according to claim 1, is characterized in that, amplifing reagent is comprised of the component with lower volume:
In above-mentioned KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, the corresponding primer of CACNB2B, described internal reference upstream primer is SEQ NO.1, and polymorphism primer is SEQ NO.2 and SEQ NO.3, and downstream primer is SEQ NO.4; The concentration of described internal reference upstream and downstream primer and polymorphism primer is 10uM; Detect the primer mixture SEQ NO.1 in KCNE1 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; Detect the primer mixture SEQ NO.1 in KCNH2 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; Detect the primer mixture SEQ NO.1 in KCNJ11 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; In the primer mixture in detection SCN5A gene polymorphic site, the consumption of SEQ NO.1 primer is 0.1ul-0.3ul, SEQ NO.2, and SEQ NO.3 and SEQ NO.4 primer consumption are respectively 0.3ul-0.7ul; Detect the primer mixture SEQ NO.1 in CACNA1C gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; The consumption that detects CACNB2B gene polymorphic site primer mixture SEQ NO.1 primer is 0.05ul-0.2ul, SEQ NO.2, and the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.6ul-1ul; The consumption that detects NUP155 gene polymorphic site primer mixture SEQ NO.1 is 0.1ul-0.3ul, SEQ NO.2, and the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul.
10. a sudden cardiac death high-speed gene test method, is characterized in that, its step comprises,
(1), the genomic DNA extraction of sample;
(2), the detection analysis of amplification and amplified production;
In above-mentioned KCNE1, KCNH2, SCN5A, KCNJ11, NUP155, CACNA1C, the corresponding primer of CACNB2B, described internal reference upstream primer is SEQ NO.1, and polymorphism primer is SEQ NO.2 and SEQ NO.3, and downstream primer is SEQ NO.4; The concentration of described internal reference upstream and downstream primer and polymorphism primer is 10uM; Detect the primer mixture SEQ NO.1 in KCNE1 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; Detect the primer mixture SEQ NO.1 in KCNH2 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; Detect the primer mixture SEQ NO.1 in KCNJ11 gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; In the primer mixture in detection SCN5A gene polymorphic site, the consumption of SEQ NO.1 primer is 0.1ul-0.3ul, SEQ NO.2, and SEQ NO.3 and SEQ NO.4 primer consumption are respectively 0.3ul-0.7ul; Detect the primer mixture SEQ NO.1 in CACNA1C gene polymorphic site, SEQ NO.2, the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul; The consumption that detects the primer mixture SEQ NO.1 primer in CACNB2B gene polymorphic site is 0.05ul-0.2ul, SEQ NO.2, and SEQ NO.3 and SEQ NO.4 consumption are respectively 0.6ul-1ul; The consumption that detects the primer mixture SEQ NO.1 primer in NUP155 gene polymorphic site is 0.1ul-0.3ul, SEQ NO.2, and the consumption of SEQ NO.3 and SEQ NO.4 is respectively 0.3ul-0.7ul;
Pcr amplification program is as follows:
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