CN103088135B - Detection kit for fluorescent PCR (Polymerase Chain Reaction) melting curve of gene mutation of glucose-6-phosphate dehydrogenase - Google Patents
Detection kit for fluorescent PCR (Polymerase Chain Reaction) melting curve of gene mutation of glucose-6-phosphate dehydrogenase Download PDFInfo
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Abstract
The invention discloses a detection kit for a fluorescent PCR (Polymerase Chain Reaction) melting curve of gene mutation of glucose-6-phosphate dehydrogenase. The kit provided by the invention can detect 16 mutations at 16 sites in a two-tube PCR system, and the genotype of a sample can be known through a primary fluorescent PCR melting curve analysis after PCR amplification. The whole operation is completed within 2-3 hours, so that the operating step is less, the time consumed is short, the flux is high, the detection sites are more and the mutation coverage is high. In addition, the kit provided by the invention uses homogeneous detection in a closed tube, so that the probability of PCR product pollution is reduced, the detection specificity is high and the result is easy to judge.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of test kit applying the sudden change of fluorescent PCR melting curve analysis detection G 6 PD gene mutations.
Background technology
Glucose 6 phosphate dehydrogenase deficiency (G6PD deficiency) is one of modal mankind's enzyme defect disease, and its cause of disease is that G6PD transgenation causes glucose-6-phosphate dehydrogenase (G6PD) to synthesize minimizing.But, therefore the quality of life of Most patients does not reduce, this is mainly owing to just there will be corresponding clinical symptom under existence only in inducement of most G6PD deficiency disease patients of occurring in China, therefore, the generation of above-mentioned situation effectively can be avoided completely by taking precautions against in advance and accept correct treatment in time.So it is very necessary for carrying out whole people's examination in the district occurred frequently of China G6PD deficiency disease.
The methods for clinical diagnosis of current G6PD deficiency disease comprises biochemical detection methods and molecular biology for detection, and its mesophytization examination is traditional diagnosis method, is come carrier and the patient of examination G6PD deficiency disease by the enzymic activity measuring G6PD directly or indirectly.Mainly comprise methemoglobin reduction test, fluorescent spot experimental tests, nitro blue tetrazolium (NBT) paper disk method of carrying out qualitative detection and carry out the NBT quantitative method, NADPH quantitative ratio method etc. of detection by quantitative.Molecular biology method is the detection carrying out G6PD transgenation based on the analysis of DNA, as reverse dot blot hybridization (RDB), PE/DHPLC, AS-PCR, PCR-SSCP, gene chip etc.Manufacturer mainly contains Niu Luoximin bio tech ltd, Hangzhou and Solgent company limited etc.Domestic clinical conventional method is detect biochemistry detection method and the revert dot blot hybridization of enzymic activity.
Although biochemical detection methods testing cost is low, detection time is short, there is following shortcoming in it: (1) recall rate to women's heterozygote is low, is only about 20 ~ 45%; (2) factors such as result is subject to experiment condition, operator affect.
Although existing molecular detecting method can solve the problem of G6PD deficiency disease women Heterozygote detation preferably, diverse ways has self limitation.Revert dot blot hybridization is a kind of mutation detection methods of out-phase, needs to carry out subsequent disposal to PCR primer, and operation steps is more, easily causes pollution, and result interpretation is subject to subjective factor impact.PE/DHPLC is high to instrument requirements equipment, and applicability is not strong.The flux that AS-PCR detects is very limited, and needs PCR aftertreatment, easily occurs polluting the generation causing false positive results.Whether PCR-SSCP is strict to requirement for experiment condition, can only detect sudden change and exist, and also needs further order-checking for the particular location suddenlyd change and type.In addition, because some point mutation type changes little, easily undetected to the space conformation of single stranded DNA.There is false positive in various degree in biochip technology not only high, the complicated operation of cost but also result.
Summary of the invention
The object of the invention is to overcome prior art defect, provide a kind of and apply the test kit that fluorescent PCR melting curve analysis detects G 6 PD gene mutations sudden change.
Technical scheme of the present invention is as follows:
A kind of G 6 PD gene mutations sudden change fluorescent PCR melting curve detection kit, this test kit comprises:
Specific amplified five kinds of mutational sites are the upstream primer F1 of place sequence and downstream primer R1 c.1360C>T, c.1376G>T, c.1381G>A, c.1387C>T and c.1388G>A, wherein, the base sequence of F1 is as shown in SEQ ID NO:1, and the base sequence of R1 is as shown in SEQ ID NO:2;
Specific amplified three kinds of mutational sites are the upstream primer F2 of place sequence and downstream primer R2 c.871G>A, c.1004C>A and c.1024C>T, wherein, the base sequence of F2 is as shown in SEQ ID NO:3, and the base sequence of R2 is as shown in SEQ ID NO:4;
Specific amplified mutational site c.95A>G the upstream primer F3 of place sequence and downstream primer R3 wherein, the base sequence of F3 is as shown in SEQ ID NO:5, and the base sequence of R3 is as shown in SEQ ID NO:6;
C.383T>C and c.392G>T specific amplified two kinds of mutational sites are the upstream primer F4 of place sequence and downstream primer R4, wherein, the base sequence of F4 is as shown in SEQ ID NO:7, and the base sequence of R4 is as shown in SEQ ID NO:8;
Specific amplified five kinds of mutational sites are the upstream primer F5 of place sequence and downstream primer R5 c.487G>A, c.493A>G, c.517T>C, c.519C>T and c.592C>T, wherein, the base sequence of F5 is as shown in SEQ ID NO:9, and the base sequence of R5 is as shown in SEQ ID NO:10;
And for detecting the fluorescent probe in above-mentioned 16 kinds of mutational sites.
In a preferred embodiment of the invention, described fluorescent probe comprises:
For detecting mutational site fluorescent probe P1 c.1360C>T, its base sequence is as shown in SEQ ID NO:11;
For detecting mutational site fluorescent probe P2 c.871G>A, its base sequence is as shown in SEQ ID NO:12;
For detecting mutational site fluorescent probe P3 c.1004C>A and c.1024C>T, its base sequence is as shown in SEQ ID NO:13;
For detecting mutational site fluorescent probe P4 c.1376G>T, c.1388G>A, c.1381G>A and c.1387C>A, its base sequence is as shown in SEQ ID NO:14;
For detecting mutational site fluorescent probe P5 c.95A>G, its base sequence is as shown in SEQ ID NO:15;
For detecting mutational site fluorescent probe P6 c.383T>C and c.392G>T, its base sequence is as shown in SEQ ID NO:16;
For detecting mutational site fluorescent probe P7 c.487G>A and c.493A>G, its base sequence is as shown in SEQ ID NO:17;
For detecting mutational site fluorescent probe P8 c.517T>C and c.519C>T, its base sequence is as shown in SEQ ID NO:18;
For detecting mutational site fluorescent probe P9 c.592C>T, its base sequence is as shown in SEQ ID NO:19.
In a preferred embodiment of the invention, the fluorophor that described fluorescent probe 5 ' is held comprises ALEX-350, FAM, VIC, TET, CAL Fluor Gold540, JOE, HEX, CAL FlourOrange560, TAMRA, Cal Fluor Red590, ROX, CAL Fluor Red610, TEXASRED, CAL Flour Red635, Quasar670, CY3, CY5, CY5.5 or Quasar705, and the quenching group that described fluorescent probe 3 ' is held comprises DABCYL, BHQ, ECLIPSE or TAMRA.
In a preferred embodiment of the invention, this test kit comprises detection system A and detection system B,
Described detection system A comprises: 1 × PCR buffer, 1U Taq archaeal dna polymerase, 200 μMs of dNTP, 2.5mM MgCl
2, each 1pmol of upstream primer F1, F2, each 5pmol of downstream primer R1, R2 and each 5pmol of fluorescent probe P1, P2, P3, P4;
Described detection system B comprises 1 × PCR buffer, 1U Taq archaeal dna polymerase, 200 μMs of dNTP, 2.5mM MgCl
2, each 1pmol of upstream primer F3, F4, F5, each 5pmol of downstream primer R3, R4, R5 and each 5pmol of fluorescent probe P5, P6, P7, P8, P9;
Above-mentioned 1 × PCR buffer comprises: Tris-HCl pH8.510mM, KCl50mM and 50%(v/v) glycerine.
In a preferred embodiment of the invention, the fluorescent PCR melting curve trace routine of described detection system A and detection system B is as follows:
（1）50℃2min，95℃10min
(2) 95 DEG C of 15s → 65 DEG C ~ 56 DEG C of 15s → 76 DEG C 20s, 10 circulations, wherein 65 DEG C ~ 56 DEG C each circulation decline of 15s 1 DEG C;
(3) 95 DEG C of 15s → 55 DEG C 15s → 76 DEG C 20s, 50 circulations, gather fluorescent signal at 55 DEG C of annealing stages;
DEG C 3min → 40, (4) 95 DEG C of 1min → 35 DEG C ~ 85 DEG C, wherein 40 DEG C ~ 85 DEG C are carried out melting curve analysis with the temperature rise rate of 0.4 DEG C/5s, and at this phase acquisition fluorescent signal.
The invention has the beneficial effects as follows:
1, easy to be quick, consuming time short: the present invention can complete the detection of 16 kinds, 16 sites sudden change in two pipe PCR system, pcr amplification terminates just can know sample genotype through first order fluorescence PCR melting curve analysis, wholely operate in 2 ~ 3 hours and can complete, operation steps is few, consuming time short;
2, homogeneous phase detection, stopped pipe operation: the present invention is homogeneous phase detection system, and PCR and melting curve analysis all complete in same closed reaction tubes, without the need to PCR aftertreatment, decrease the possibility that PCR primer is polluted;
3, detection site is many, sudden change fraction of coverage is high: the present invention can detect 16 kinds of sudden changes in G6PD gene 16 sites simultaneously, and detection site is many, high to the sudden change fraction of coverage of Chinese population;
4, flux is detected high: the present invention is based on fluorescent PCR melting curve analysis technology, only need run a simple melting curve analysis step (completing within 40min on fluorescent PCR instrument) after PCR can complete, and PCR can run on General Instrument, a fluorescent PCR instrument can coordinate multiple stage regular-PCR instrument to complete melting curve analysis, therefore can greatly improve detection flux, improve the utilization ratio of fluorescent PCR instrument;
5, high, the easy interpretation of result of detection specificity: the present invention is that the fusing point by melting peak judges whether to suddenly change, and result is easy to interpretation, and not easily makes mistakes, therefore detection specificity is high.
Accompanying drawing explanation
Fig. 1 is the detected result figure of the FAM fluorescence channel of the detection system A of test kit of the present invention in embodiment 2;
Fig. 2 is the detected result figure of the HEX fluorescence channel of the detection system A of test kit of the present invention in embodiment 2;
Fig. 3 is the detected result figure of the ROX fluorescence channel of the detection system A of test kit of the present invention in embodiment 2;
Fig. 4 is the detected result figure of the Cy5 fluorescence channel of the detection system A of test kit of the present invention in embodiment 2;
Fig. 5 is the detected result figure of the FAM fluorescence channel of the detection system B of test kit of the present invention in embodiment 2;
Fig. 6 is the detected result figure of the HEX fluorescence channel of the detection system B of test kit of the present invention in embodiment 2;
Fig. 7 is the detected result figure of the ROX fluorescence channel of the detection system B of test kit of the present invention in embodiment 2;
Fig. 8 is the detected result figure of the Cy5 fluorescence channel of the detection system B of test kit of the present invention in embodiment 2.
Embodiment
Will by reference to the accompanying drawings below by way of embodiment, technical scheme of the present invention is further detailed and is described.
Embodiment 1
Primer in this test kit and probe are according to the G 6 PD gene mutations sequence in Genebank and mutational site design thereof, specific as follows:
F1：5’-GGCATGTTCTTCAACC-3’（SEQ?ID?NO：1），
R1：5’-GCCCTCATACTGGAAA-3’（SEQ?ID?NO：2），
F1/R1 specific amplified five kinds of mutational sites c.1360C>T, c.1376G>T, c.1381G>A, c.1387C>T and c.1388G>A place sequence;
F2：5’-AGAAGTCACCACCTCT-3’（SEQ?ID?NO：3），
R2：5’-GGCCACATCATGGAAC-3’（SEQ?ID?NO：4），
F2/R2 specific amplified three kinds of mutational sites c.871G>A, c.1004C>A and c.1024C>T place sequence;
F3：5’-TTTACTCGGTTGTCCAGAA-3’（SEQ?ID?NO：5），
R3：5’-GGCGACCAGAGCAAA-3’（SEQ?ID?NO：6），
F3/R3 specific amplified mutational site c.95A>G place sequence;
F4：5’-TGCTCTCGTACTTCCTT-3’（SEQ?ID?NO：7），
R4：5’-TTGAAAATGAGAAGAGGACC-3’（SEQ?ID?NO：8），
F4/R4 specific amplified two kinds of mutational sites c.383T>C and c.392G>T place sequence;
F5：5’-GAGGAAGCCATGGCTT-3’（SEQ?ID?NO：9），
R5：5’-AAACCGTGGGGTGCTT-3’（SEQ?ID?NO：10），
Specific amplified five kinds of mutational sites c.487G>A, c.493A>G, c.517T>C, c.519C>T and c.592C>T place sequence;
C.1360C>T P1:5 '-FAM-GGCCAGATGCACTTCGTGCGTGGCC-BHQ1-3 ' (SEQ IDNO:11), for detecting mutational site;
C.871G>A P2:5 '-HEX-AGCCGGTGTTGAAATGCATCTCAGAGGGCT-BHQ1-3 ' (SEQ ID NO:12), for detecting mutational site;
C.1004C>A and c.1024C>T P3:5 '-ROX-GGCCACTTTTGCAGCCGTCGTCTGTGGCC-BHQ2-3 ' (SEQ ID NO:13), for detecting mutational site;
C.1376G>T, c.1388G>A, c.1381G>A and c.1387C>A P4:5 '-CY5-GGACCTCCTTGAGGCCTGGCGTGGTCC-BHQ2-3 ' (SEQID NO:14), for detecting mutational site;
C.95A>G P5:5 '-FAM-GCGGCATATTCATCATCATGGGTGCCGC-BHQ1-3 ' (SEQID NO:15), for detecting mutational site;
C.383T>C and c.392G>T P6:5 '-FAM-GGCTCCACCTGGGGTCACAGGCGGAGCC-BHQ1-3 ' (SEQID NO:16), for detecting mutational site;
P7：
C.487G>A and c.493A>G 5 '-HEX-GCGCCAAGCTGGAACCGCATCATCGTGGAGAATGGCGC-BHQ1-3 ' (SEQ ID NO:17), for detecting mutational site;
C.517T>C and c.519C>T P8:5 '-ROX-GGGTTCGGGAGGGACCTGCAGAGCTAACCC-BHQ2-3 ' (SEQ ID NO:18), for detecting mutational site;
C.592C>T P9:5 '-CY5-GGCCACGCATCGACCACTACCTGGGTGGCC-BHQ2-3 ' (SEQ ID NO:19), for detecting mutational site.
Detection system in this test kit comprises detection system A and detection system B,
Described detection system A comprises: 1 × PCR buffer, 1U Taq archaeal dna polymerase, 200 μMs of dNTP, 2.5mM MgCl
2, each 1pmol of upstream primer F1, F2, each 5pmol of downstream primer R1, R2 and each 5pmol of fluorescent probe P1, P2, P3, P4;
Described detection system B comprises 1 × PCR buffer, 1U Taq archaeal dna polymerase, 200 μMs of dNTP, 2.5mM MgCl
2, each 1pmol of upstream primer F3, F4, F5, each 5pmol of downstream primer R3, R4, R5 and each 5pmol of fluorescent probe P5, P6, P7, P8, P9;
Above-mentioned 1 × PCR buffer comprises: Tris-HCl pH8.510mM, KCl50mM and 50%(v/v) glycerine.
The fluorescent PCR melting curve trace routine of above-mentioned detection system A and detection system B is as follows:
（1）50℃2min，95℃10min
(2) 95 DEG C of 15s → 65 DEG C ~ 56 DEG C of 15s → 76 DEG C 20s, 10 circulations, wherein 65 DEG C ~ 56 DEG C each circulation decline of 15s 1 DEG C;
(3) 95 DEG C of 15s → 55 DEG C 15s → 76 DEG C 20s, 50 circulations, gather FAM, HEX, ROX and CY5 fluorescent signal at 55 DEG C of annealing stages;
DEG C 3min → 40, (4) 95 DEG C of 1min → 35 DEG C ~ 85 DEG C, wherein 40 DEG C ~ 85 DEG C are carried out melting curve analysis with the temperature rise rate of 0.4 DEG C/5s, and at this phase acquisition FAM, HEX, ROX and CY5 fluorescent signal.
Embodiment 2
The test kit of Application Example 1 carries out detection and analyzes, and the instrument that operating process uses is Bio-RadCFX96 real-time fluorescence PCR instrument:
(1) to increase respectively with 5 pairs of primers in the test kit of embodiment 1 16 mutational sites of glucose-6-phosphate dehydrogenase (G6PD), 16 mutant nucleotide sequences are obtained with this, the PCR primer of gained 16 mutant nucleotide sequences is inserted in pMD18-T plasmid vector respectively, obtains 16 sudden change positive criteria product with this;
(2) 16 that obtain in step (1) sudden change positive criteria product, glucose-6-phosphate dehydrogenase (G6PD) wild-type (Wt) sequence and negative controls (NTC) are joined in the detection system of the test kit of embodiment 1 respectively, and detect by the fluorescent PCR melting curve trace routine of the test kit of embodiment 1, the detection system A of each sample and the template add-on of detection system B are 5 microlitres, every microlitre 3 × 10
3copy; Negative control is 10mM Tris-EDTA damping fluid, pH8.0, and add-on is 5 microlitres.
(3) result interpretation: in step (2), often kind of genotypic sample of glucose-6-phosphate dehydrogenase has its feature Tm value (without melting curve detection signal in negative control) in different fluorescence detection channel, calculate wild type control in the Tm value of each passage and 16 differences of positive criteria product in the Tm value of each passage of suddenling change, i.e. Δ Tm value, is summarized as follows table by the relation of gained Δ Tm value and fluorescent probe and sense channel:
As shown in Figures 1 to 8, test kit of the present invention all can detect special melting curve detection signal to above-mentioned 16 kinds of glucose-6-phosphate dehydrogenase mutation types, and between each genotype, there is not the phenomenon of intersecting and detecting, the genotype of the Δ Tm value interpretation testing sample in table therefore can be contrasted when detecting actual sample.
The above, be only preferred embodiment of the present invention, therefore can not limit scope of the invention process according to this, the equivalence change namely done according to the scope of the claims of the present invention and description with modify, all should still belong in scope that the present invention contains.
Claims (2)
1. a G 6 PD gene mutations sudden change fluorescent PCR melting curve detection kit, it is characterized in that: this test kit comprises: specific amplified five kinds of mutational sites are the upstream primer F1 of place sequence and downstream primer R1 c.1360C>T, c.1376G>T, c.1381G>A, c.1387C>T and c.1388G>A, wherein, the base sequence of F1 is as shown in SEQ ID NO:1, and the base sequence of R1 is as shown in SEQ ID NO:2; Specific amplified three kinds of mutational sites are the upstream primer F2 of place sequence and downstream primer R2 c.871G>A, c.1004C>A and c.1024C>T, wherein, the base sequence of F2 is as shown in SEQ ID NO:3, and the base sequence of R2 is as shown in SEQ ID NO:4; Specific amplified mutational site c.95A>G the upstream primer F3 of place sequence and downstream primer R3 wherein, the base sequence of F3 is as shown in SEQ ID NO:5, and the base sequence of R3 is as shown in SEQ ID NO:6; C.383T>C and c.392G>T specific amplified two kinds of mutational sites are the upstream primer F4 of place sequence and downstream primer R4, wherein, the base sequence of F4 is as shown in SEQ ID NO:7, and the base sequence of R4 is as shown in SEQ ID NO:8; Specific amplified five kinds of mutational sites are the upstream primer F5 of place sequence and downstream primer R5 c.487G>A, c.493A>G, c.517T>C, c.519C>T and c.592C>T, wherein, the base sequence of F5 is as shown in SEQ ID NO:9, and the base sequence of R5 is as shown in SEQ ID NO:10;
And for detecting the fluorescent probe in above-mentioned 16 kinds of mutational sites, described fluorescent probe comprises: for detecting mutational site fluorescent probe P1 c.1360C>T, its base sequence is as shown in SEQ ID NO:11; For detecting mutational site fluorescent probe P2 c.871G>A, its base sequence is as shown in SEQ ID NO:12; For detecting mutational site fluorescent probe P3 c.1004C>A and c.1024C>T, its base sequence is as shown in SEQ ID NO:13; For detecting mutational site fluorescent probe P4 c.1376G>T, c.1388G>A, c.1381G>A and c.1387C>A, its base sequence is as shown in SEQ ID NO:14; For detecting mutational site fluorescent probe P5 c.95A>G, its base sequence is as shown in SEQ ID NO:15; For detecting mutational site fluorescent probe P6 c.383T>C and c.392G>T, its base sequence is as shown in SEQ ID NO:16; For detecting mutational site fluorescent probe P7 c.487G>A and c.493A>G, its base sequence is as shown in SEQ ID NO:17; For detecting mutational site fluorescent probe P8 c.517T>C and c.519C>T, its base sequence is as shown in SEQ ID NO:18; For detecting mutational site fluorescent probe P9 c.592C>T, its base sequence is as shown in SEQ ID NO:19;
The fluorophor that described fluorescent probe 5 ' is held comprises ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Flour Orange 560, TAMRA, Cal Fluor Red590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Flour Red 635, Quasar 670, CY3, CY5, CY5.5 or Quasar 705, and the quenching group that described fluorescent probe 3 ' is held comprises DABCYL, BHQ, ECLIPSE or TAMRA;
This test kit comprises detection system A and detection system B,
Described detection system A comprises: 1 × PCR buffer, 1U Taq archaeal dna polymerase, 200 μMs of dNTP, 2.5mM MgCl
2, each 1 pmol of upstream primer F1, F2, each 5 pmol of downstream primer R1, R2 and each 5 pmol of fluorescent probe P1, P2, P3, P4;
Described detection system B comprises 1 × PCR buffer, 1U Taq archaeal dna polymerase, 200 μMs of dNTP, 2.5mM MgCl
2, each 1 pmol of upstream primer F3, F4, F5, each 5pmol of downstream primer R3, R4, R5 and each 5 pmol of fluorescent probe P5, P6, P7, P8, P9;
Above-mentioned 1 × PCR buffer comprises: Tris-HCl pH8.5 10mM, KCl 50mM and 50% (v/v) glycerine.
2. a kind of G 6 PD gene mutations sudden change fluorescent PCR melting curve detection kit as claimed in claim 1, is characterized in that: the fluorescent PCR melting curve trace routine of described detection system A and detection system B is as follows:
(1)50℃2min，95℃10min；
(2) 95 DEG C of 15s → 65 DEG C ~ 56 DEG C of 15s → 76 DEG C 20s, 10 circulations, wherein 65 DEG C ~ 56 DEG C each circulation decline of 15s 1 DEG C;
(3) 95 DEG C of 15s → 55 DEG C 15s → 76 DEG C 20s, 50 circulations, gather fluorescent signal at 55 DEG C of annealing stages;
DEG C 3min → 40, (4) 95 DEG C of 1min → 35 DEG C ~ 85 DEG C, wherein 40 DEG C ~ 85 DEG C are carried out melting curve analysis with the temperature rise rate of 0.4 DEG C/5s, and at this phase acquisition fluorescent signal.
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| CN111118141B (en) * | 2020-01-15 | 2023-09-01 | 浙江迪谱诊断技术有限公司 | Primer sequence and kit for detecting glucose-6-phosphate dehydrogenase (G6 PD) gene mutation |
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| CN101608215A (en) * | 2008-06-19 | 2009-12-23 | 上海裕隆生物科技有限公司 | A kind of genotype tests method of combined with fluorescent quantitative PCR |
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