CN105506068A - AMRMS-qPCR detection kit and detection method for MMP2 genotyping - Google Patents
AMRMS-qPCR detection kit and detection method for MMP2 genotyping Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to an AMRMS-qPCR detection kit for MMP2 genotyping and an AMRMS-qPCR detection method, based on the detection kit, for MMP2 genotyping. The detection kit comprises a qPCR reaction system. The qPCR reaction system comprises qPCR mixed reaction liquid, a reference primer, an ARMS primer and a positive control sample. The qPCR mixed reaction liquid comprises PCR buffer solution, dNTP, MgCl2, Tag enzyme, a PCR downstream primer and a TaqMan probe. In the qPCR reaction system, the final concentration of the PCR buffer solution is 1X, the final concentration of the dNTP is 0.1-1.5mM, the final concentration of the MgCl2 is 0.5-5mM, the final concentration of the Tag enzyme is 0.05-0.1U/microliter, the final concentration of the PCR downstream primer is 0.1-1microM, the final concentration of the TaqMan probe is 0.1-1microM, the final concentration of the reference primer is 0.5microM, the final concentration of the ARMS primer is 0.5microM and the final concentration of the positive control sample is 1-10ng/microliter. Compared with genotyping technologies such as direct sequencing, the AMRMS-qPCR detection kit for MMP2 genotyping is high in specificity, high in sensitivity, simple and fast to operate, high in throughput, reliable in detection result, simple, and the like.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of ARMS-qPCR detection kit for MMP2 gene type, the invention still further relates to a kind of MMP2 gene type ARMS-qPCR detection method.
Background technology
Obesity is a kind of chronic disease, estimates according to the World Health Organization, and it is the most out in the cold to be that the mankind face at present, but a kind of disease that sickness rate is sharply rising.Obesity can cause multiple physiology and mental illness, such as diabetes, cancer, cardiovascular disorder and human communication disorders etc.Cause the reason of obesity a lot, such as excessive Energy intaking and the mode of life etc. of silent oscillation.Increasing research shows, inherited genetic factors plays important role (Fraylingetal.2007, Gerkenetal.2007, Dinaetal.2007, Chuetal.2008) in obesity pathogenic process.
MMP2 gene is positioned at No. 16 karyomit(e), a matrix metalloproteinase of encoding, and major function is for shearing extracellular matrix.Recently, studies have found that MMP2rs1132896 genotype and women obesity disease tight association (Bouwan and Boer2013).Rs1132896CC type has very high obesity probability of occurrence relative to rs1132896CT type and TT type.Therefore the genotype that rs1132896 is different is a fabulous women obesity disease risk profile index, has important directive significance for the prevention of obesity and the treatment in its later stage.
The technology that can be used for MMP2rs1132896 somatotype is a lot, comprises as DNA direct sequencing, hybridization hybrid chip method, Manganic pyrophosphate complex initiation method and denaturing high-performance liquid chromatography etc.Wherein, DNA sequencing method is the gold standard of gene type, and the method exists following shortcoming: the sensitivity of detection is not high, and the sudden change being only greater than 15% just can detect; Time-consuming, complicated operation, requires high to operator; Non-stopped pipe operation, relates to the operation after pcr amplification, therefore easily contaminated, causes the undesirable of result; Flux is too little, once can only do 24 at most, is difficult to carry out on a large scale.
Summary of the invention
Technical problem to be solved by this invention is the ARMS-qPCR detection kit for MMP2 gene type providing a kind of highly sensitive, high specificity for the state of the art.
Second technical problem to be solved by this invention is to provide a kind of ARMS-qPCR detection method of MMP2 gene type, and this detection method is highly sensitive, high specificity, and simple to operate.
The present invention solves the technical scheme that above-mentioned first technical problem adopt: a kind of ARMS-qPCR detection kit for MMP2 gene type, comprise qPCR reaction system, described qPCR reaction system comprises qPCR mixed reaction solution, with reference to primer, ARMS primer and positive controls sample; Described qPCR mixed reaction solution comprises PCR damping fluid, dNTP, MgCl
2, Tag enzyme, PCR downstream primer and TaqMan probe;
In described qPCR reaction system, the final concentration of each component is respectively: PCR damping fluid is 1X; DNTP is 0.1 ~ 1.5mM; MgCl
2be 0.5 ~ 5mM; Tag enzyme is 0.05 ~ 0.1U/ μ l; PCR downstream primer is 0.1 ~ 1 μM; TaqMan probe is 0.1 ~ 1 μM; Reference primer is 0.5 μM; ARMS primer is 0.5 μM; Positive control sample is 1 ~ 10ng/ μ l.
The sequence of described PCR downstream primer is as shown in MMP2-R;
The sequence of described TaqMan probe is as shown in MMP2-TaqMan;
The described sequence with reference to primer is as shown in MMP2-C, and can increase all different MMP2 genotype;
Described ARMS primer is any one in two kinds of upstream primers, respectively corresponding MMP2CCrs1132896, TTrs1132896 type genotype, and the sequence of these two kinds of upstream primers is as shown in MMP2-F1, MMP2-F2; These two kinds of ARMS primers, respectively at 3 ' end and different genotype Mismatchings, increase one or two base mispairings, to increase its specificity 2nd, 3 reciprocal of its 3 ' end simultaneously; MMP2rs1132896 position genotype for specific amplification is CC/TT/TC;
Described positive controls sample is respectively the plasmon DNA of MMP2 different genotype base substitution.
Wherein, described Tag enzyme is TakaraTag enzyme or GoldstarBestTag enzyme.
Each primer sequence is as follows:
MMP2-R：5’-TCTTGGGGACTAGAGTGGAT-3’
MMP2-TaqMan：5’-TGCACTGATACCGGCCGCAG-3’
MMP2-C：5’-TCCAACCTCCCCCTTCCATG-3’
MMP2-F1：5’-TGGTCCGTGTGAAGTATCAC-3’
MMP2-F2：5’-TGGTCCGTGTGAAGTATCAG-3’
The present invention solves the technical scheme that above-mentioned second technical problem adopt: based on the MMP2 gene type ARMS-qPCR detection method of mentioned reagent box, the method before this for MMP2 gene design a pair with reference to primer, TaqMan probe, for the different genotype design ARMS primer in rs1132896 site, qPCR mixed reaction solution is added in reaction system, carry out the detection of quantitative fluorescent PCR with reference to primer or ARMS primer, TaqMan primer and template DNA to be measured, specifically comprise the steps:
(1) design and screen containing general reference primer, TaqMan probe and the two kinds of ARMS primers of MMP2 gene rs1132896 position different genotype;
(2) from cell system or blood, genomic dna is extracted as template DNA;
(3) real-time fluorescence quantitative PCR amplification is carried out, the detection of each sample divides 3 pipes to carry out, each pipe adds identical qPCR mixed reaction solution, TaqMan probe and template DNA, often pipe adds respectively with reference to the one in primer and two kinds of ARMS primers, and the qPCR carrying out MMP2 gene rs1132896 position gene type detects;
(4) result interpretation:
Positive with reference to primer pipe amplification curve, MMP2-F1 pipe amplification curve is positive, the amplification curve Δ CT > 10 that MMP2-F2 pipe amplification curve is negative or it is with reference primer pipe, and this sample results is judged to be rs1132896CC genotype;
Positive with reference to primer pipe amplification curve, MMP2-F1 pipe amplification curve is positive, and MMP2-F2 pipe amplification curve is positive, and this sample results is judged to be rs1132896C/T genotype;
Positive with reference to primer pipe amplification curve, MMP2-F1 pipe amplification curve negative or itself and amplification curve Δ CT > 10, the MMP2-F2 pipe amplification curve positive with reference to primer pipe, this sample results is judged to be rs1132896TT genotype;
Negative with reference to primer pipe amplification curve, point out this sample extraction failure, need to re-start extraction.
In described step (2), template DNA is selected from blood of human body genomic dna or oral mucosa cast-off cells or hair.
In described step (3), the condition of carrying out pcr amplification reaction is, 95 DEG C of denaturation 2min, 95 DEG C of sex change 15s, and 60 DEG C extend 1min, 46 circulations.
The Full Name in English of qPCR mentioned in technique scheme is Real-timeQuantitativePCRDetectingSystem.I.e. real time fluorescent quantitative nucleic acid amplification detection system, is also real-time quantitative gene amplification fluorescence detecting system, is called for short qPCR.
Compared with prior art, the invention has the advantages that:
The invention provides a kind of ARMS-qPCR detection kit of MMP2 gene type, with solve time-consuming in existing genotype tests technology, program is loaded down with trivial details and the problem such as easy pollution.This test kit can fast, accurately, cheap, high-throughput carries out somatotype to MMP2 gene, and it is highly sensitive, high specificity, is applicable to common sample such as blood, oral mucosa and hair etc.
The present invention can be quick, cheap, accurate, high-throughput ground detects the type that in human body blood or other histocytes, MMP2 gene is different, for typing method, be current cost performance the best way, be convenient to clinical or health detection and carry out on a large scale.
ARMS (amplificationrefractorymutationsystem) the i.e. amplification refractory mutation system that the present invention is used, for detecting known mutations gene.This method is by design two 5 ' end primer, one complementary with normal DNA, and one complementary with mutant DNA, for homozygous mutant, adding these two kinds of primers and 3 ' respectively holds primer to carry out two parallel PCR, only has the primer of the complete complementation with mutant DNA just extensible and obtains pcr amplification product.If mispairing is positioned at 3 ' end of primer, causes PCR not extend, be then called ARMS.
The sensitivity that ARMS detects depends on the specificity of ARMS primer and reaction conditions as the concentration of MgCl2 in reaction solution, adds the optimization of DMSO etc.In order to increase the specificity of primer, the mispairing reducing primer and the wrong timing of target DNA extends, by primer 3, ' the 2nd, 3 base of end introduces another one or two base mismatch, makes it to form multiple mispairing between template and extends with IMPEDANCE WRONG.
The present invention's probe used is the TaqMan probe of 5 ' end FAM mark, and probe two ends are mark fluorescent reporter group (R) and fluorescent quenching group (Q) respectively.When probe is complete, time namely in random state with without PCR primer hybridized state, the fluorescence that reporter group sends is quenched group absorptions, can't detect the existence of fluorescence.In ARMS-qPCR amplification procedure, when special PCR primer and TaqMan probe generation hybridization, 5 ' end 5 prime excision enzyme activity of GoldstarBestTag enzyme is also sheared the base of TaqMan probe one by one, and the fluorescence that reporter group discharges the just photofluorometer that can be built in PCR instrument detects.PCR is through a circulation, and fluorescent signal is also the same with object fragment, has the process of sync index amplification, the power of fluorescent signal just represent template DNA copy number number.Therefore the present invention is an instrument for good gene type.
Combine the advantage of ARMS primer and real-time fluorescence quantitative PCR, compared with the technology of the gene type such as direct Sequencing, the present invention is used for MMP2 genotyping gene and detects and has following advantage:
1, high specificity: the ARMS primer of design is respectively for the special series of variation of MMP2TTrs1132896, CCrs1132896, and can increase corresponding genotype DNA specifically; Inverse the 2nd, 3 bases of holding at ARMS primer 3 ' introduce another one or two base mismatch, increase the specificity of ARMS primer;
2, the sensitivity of the technology of the present invention can reach 1%, far above the sensitivity of DNA direct Sequencing 15%;
3, testing process is stopped pipe reaction, greatly reduces the possibility of pollution and result error;
4, operation is simple fast, can complete from sample censorship to obtaining result in 3 hours.And direct sequencing detecting step is loaded down with trivial details: censorship sample → extraction DNA → pcr amplification → checking PCR primer (electrophoresis) → purified pcr product → direct Sequencing → interpretation of result, it is through the electrophoresis process of two PCR primer, pollute probability very large, be not suitable for carrying out on a large scale in hospital;
5, sentence read result is clearly objective, can carry out quantitative analysis time if desired to result;
6, high-throughput, once can detect 96 samples;
7, safety, does not comprise hazardous and noxious substances in whole system, without the need to the aftertreatment of PCR primer, to operator and environment all harmless.
In a word, compared with the technology of the gene type such as direct Sequencing, the present invention is used for MMP2 gene type high specificity, highly sensitive, simple to operate fast, the advantage such as high-throughput, sentence read result be reliable, easy.
Accompanying drawing explanation
Fig. 1 is for detect a genotypic amplification curve in patients with depression MMP2rs1132896 position with MMP2ARMS-qPCR genotyping kit, and after testing, the genotype of this patient in rs1132896 position is MMP2CCrs1132896;
Fig. 2 is for detecting the genotypic amplification curve in normal people MMP2rs1132896 position with MMP2ARMS-qPCR genotyping kit, and after testing, the genotype of this patient in rs1132896 position is MMP2TTrs1132896;
Fig. 3 is for detecting the genotypic amplification curve in normal people MMP2rs1132896 position with MMP2ARMS-qPCR genotyping kit, and after testing, the genotype of this patient in rs1132896 position is MMP2C/Trs1132896.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Collect the blood sample of 100 routine patients with depression and 50 routine normal peoples, by the method for ARMS-qPCR, somatotype is carried out to its MMP2 gene rs1132896 position.
Design and screen the above-mentioned 3 kinds of genotypic ARMS primers of energy specific detection each one and reference primer one, design general downstream primer one, design and screen TaqMan general probe one, the sequence of each primer, probe is as follows:
MMP2-R：5’-TCTTGGGGACTAGAGTGGAT-3’
MMP2-TaqMan：5’-TGCACTGATACCGGCCGCAG-3’
MMP2-C：5’-TCCAACCTCCCCCTTCCATG-3’
MMP2-F1：5’-TGGTCCGTGTGAAGTATCAC-3’
MMP2-F2：5’-TGGTCCGTGTGAAGTATCAG-3’
The optimization of reaction system:
(1) optimization of primer concentration, in reaction system, other conditions are identical, primer concentration is done multiple proportions serial dilution from 0.1 μM/L ~ 1.5 μM/L, and the analysis of result determines that optimum concn is 0.5 μM/L by experiment.
(2) optimization of concentration and probe concentration, in reaction system, other conditions are identical, primer concentration is done multiple proportions serial dilution from 0.05 μM/L ~ 0.5 μM/L, and the analysis of result determines that optimum concn is 0.1 μM/L by experiment.
(3) optimization of annealing temperature, other conditions are identical in reaction system, carry out grads PCR (55 DEG C ~ 65 DEG C), the analysis of result determines that optimum temps is 60 DEG C by experiment.
(4) optimization of amplified reaction Tag enzyme, by comparing the various pcr amplification Tag enzymes on market, chooses GoldstarBestTag enzyme.
(5) after optimizing, final reaction system is 25 μ l, comprise qPCR mixed reaction solution 23 μ l, with reference to primer and each 0.5 μ l of ARMS primer (final concentration 0.5 μM/L), template DNA 1 μ l (final concentration 1-10ng/ μ l), qPCRmix (i.e. qPCR mixed reaction solution) component and final concentration thereof are:
ARMS-qPCR detects each sample and carries out 3 tube reactions on ABI7900 detector, and often pipe adds identical qPCRmix, general downstream primer, TaqMan probe, and each pipe difference is with reference to primer or ARMS primer.PCR reaction conditions is: 95 DEG C of denaturation 10min, 95 DEG C of 15s, 60 DEG C of 1min, and amplified reaction is 46 circulations.
Result is: have 82 people in 100 routine women obesity disease patients for MMP2 gene C Crs1132896 genotype, 11 people are T/Crs1132896, and 7 people are CCrs1132896 genotype; Have 30 people in 50 people's normal posture women populations for TTrs1132896 type, 13 people are T/Crs1132896 genotype, and 7 people are CCrs1132896 genotype, through the checking of DNA direct Sequencing, completely consistent with ARMS-qPCR result.
The above results shows, MMP2 Gene A RMS-qPCR classifying method method of the present invention is reliable, and highly sensitive, simple to operate, sentence read result is objective, is beneficial to larger scale clinical and carries out.
Above content is only preferred embodiment of the present invention, and for those of ordinary skill in the art, according to thought of the present invention, all will change in specific embodiments and applications, this description should not be construed as limitation of the present invention.
Claims (5)
1. for an ARMS-qPCR detection kit for MMP2 gene type, it is characterized in that: comprise qPCR reaction system, described qPCR reaction system comprises qPCR mixed reaction solution, with reference to primer, ARMS primer and positive controls sample; Described qPCR mixed reaction solution comprises PCR damping fluid, dNTP, MgCl
2, Tag enzyme, PCR downstream primer and TaqMan probe;
In described qPCR reaction system, the final concentration of each component is respectively: PCR damping fluid is 1X; DNTP is 0.1 ~ 1.5mM; MgCl
2be 0.5 ~ 5mM; Tag enzyme is 0.05 ~ 0.1U/ μ l; PCR downstream primer is 0.1 ~ 1 μM; TaqMan probe is 0.1 ~ 1 μM; Reference primer is 0.5 μM; ARMS primer is 0.5 μM; Positive control sample is 1 ~ 10ng/ μ l.
The sequence of described PCR downstream primer is as shown in MMP2-R;
The sequence of described TaqMan probe is as shown in MMP2-TaqMan;
The described sequence with reference to primer is as shown in MMP2-C, and can increase all different MMP2 genotype;
Described ARMS primer is any one in two kinds of upstream primers, respectively corresponding MMP2CCrs1132896, TTrs1132896 type genotype, and the sequence of these two kinds of upstream primers is as shown in MMP2-F1, MMP2-F2; These two kinds of ARMS primers are respectively at 3 ' end and different genotype Mismatchings, and increase one or two base mispairings 2nd, 3 reciprocal of its 3 ' end, the MMP2rs1132896 position genotype for specific amplification is CC/TT/TC simultaneously;
Described positive controls sample is respectively the plasmon DNA of MMP2 different genotype base substitution.
2. the ARMS-qPCR detection kit for MMP2 gene type according to claim 1, is characterized in that: described Tag enzyme is TakaraTag enzyme or GoldstarBestTag enzyme.
3., based on the MMP2 gene type ARMS-qPCR detection method of test kit described in claim 1 or 2, it is characterized in that, comprise the steps:
(1) design and screen containing general reference primer, TaqMan probe and the two kinds of ARMS primers of MMP2 gene rs1132896 position different genotype;
(2) from cell system or blood, genomic dna is extracted as template DNA;
(3) real-time fluorescence quantitative PCR amplification is carried out, the detection of each sample divides 3 pipes to carry out, each pipe adds identical qPCR mixed reaction solution, TaqMan probe and template DNA, often pipe adds respectively with reference to the one in primer and two kinds of ARMS primers, and the qPCR carrying out MMP2 gene rs1132896 position gene type detects;
(4) result interpretation:
Positive with reference to primer pipe amplification curve, MMP2-F1 pipe amplification curve is positive, the amplification curve Δ CT > 10 that MMP2-F2 pipe amplification curve is negative or it is with reference primer pipe, and this sample results is judged to be rs1132896CC genotype;
Positive with reference to primer pipe amplification curve, MMP2-F1 pipe amplification curve is positive, and MMP2-F2 pipe amplification curve is positive, and this sample results is judged to be rs1132896C/T genotype;
Positive with reference to primer pipe amplification curve, MMP2-F1 pipe amplification curve negative or itself and amplification curve Δ CT > 10, the MMP2-F2 pipe amplification curve positive with reference to primer pipe, this sample results is judged to be rs1132896TT genotype;
Negative with reference to primer pipe amplification curve, point out this sample extraction failure, need to re-start extraction.
4. detection method according to claim 3, is characterized in that: in described step (2), and template DNA is selected from blood of human body genomic dna or oral mucosa cast-off cells or hair.
5. detection method according to claim 3, is characterized in that: in described step (3), and the condition of carrying out pcr amplification reaction is, 95 DEG C of denaturation 2min, 95 DEG C of sex change 15s, and 60 DEG C extend 1min, 46 circulations.
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| CN110643689A (en) * | 2019-10-29 | 2020-01-03 | 陕西佰美基因股份有限公司 | TaqMan probe real-time fluorescent PCR method for detecting rs6313 site of HTR2A gene and primer probe combination thereof |
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| CN108315396A (en) * | 2018-03-28 | 2018-07-24 | 潍坊兴旺生物种业有限公司 | A kind of new method of simple and convenient detection SNP |
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| CN109971859A (en) * | 2019-04-29 | 2019-07-05 | 凯杰（苏州）转化医学研究有限公司 | A kind of detection primer and its application of abdominal aneurvsm associated SNP positions rs10757278 |
| CN110643689A (en) * | 2019-10-29 | 2020-01-03 | 陕西佰美基因股份有限公司 | TaqMan probe real-time fluorescent PCR method for detecting rs6313 site of HTR2A gene and primer probe combination thereof |
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