CN102851366A - Fluorescence quantitative PCR detection kit for beta-thalassemia mutation - Google Patents
Fluorescence quantitative PCR detection kit for beta-thalassemia mutation Download PDFInfo
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Abstract
The invention relates to a fluorescence quantitative PCR detection kit for beta-thalassemia mutation. The kit comprises a PCR mixing reaction liquid, a positive control and fluorescence probes for detecting beta-thalassemia mutation genotype, wherein the PCR mixing reaction liquid contains PCR primers for amplifying a gene fragment on which a mutation site is positioned, and the mutation site is at least one mutation site selected from deletion mutation of a base corresponding to the site 41/42 amino acid of beta-globin gene, C-to-T mutation of a base corresponding to the site 654 amino acid of the second intron of beta-globin gene, A-to-T mutation of a base corresponding to the site 17 amino acid of beta-globin gene, A-to-G mutation of a base corresponding to the site 28 amino acid on the upstream of the promoter of beta-globin gene, A base insertion mutation of a base corresponding to the site 71/72 amino acid of beta-globin gene, and G-to-C mutation of a base corresponding to the site 5 amino acid of the first intron of beta-globin gene. With the technical scheme of the present invention, rapid, accurate and high sensitivity detection of mutation conditions of beta-thalassemia gene can be achieved, and especially 6 special site mutations of beta-thalassemia gene can be detected, wherein the 6 special site mutations are common in Chinese.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of PCR kit for fluorescence quantitative that detects transgenation, relate in particular to a kind of β-thalassemia sudden change fluorescent quantificationally PCR detecting kit.
Background technology
Thalassemia (thalassem ia) is called for short " poor ", is one of modal blood inherited disease in the world.The thalassemia clinical phenotypes is various, by light to heavy, can be from normally to Transfusion, even threat to life, cause the patient namely to die young even fail before being carrying out teenage, give family and socially bring huge spirit and economical load.And, medically there is no at present effective treatment measure; Therefore, the natality of the poor infant of control and reduction ground could be monitored the generation of this type of disease effectively.By Mass screening and genetic counseling, those pregnant fetuses of couple at child-bearing age institute that carry the poor gene in ground are implemented antenatal diagnosis and gene diagnosis, be the most effective approach of the poor infant birth in control severe ground under the current conditions.
Thalassemia is from body recessive inheritance disease, because the dyssynthesis of globin chain produces, shows as the less and lost of life of erythrocyte volume.Usually the kind according to globin chain is divided into thalassemia the types such as α, β, γ, δ β, and is wherein common with α, beta Thalassemia, and beta Thalassemia harm is maximum.Wherein, β-thalassemia is a kind of hereditary hemolytic anemia take the synthetic defective of globin skin chain as feature, is mainly beta globin genes point mutation, little disappearance or insertion.β-thalassemia that the present whole world has been found is suddenlyd change about 170 kinds, has found 21 kinds in Chinese.A large amount of results of study shows that β-thalassemia transgenation and frequency distribution have obvious racial traits and areal variation.Medical statistics finds that the common sudden change of Chinese is followed successively by: sudden change (8%), the CD71-72(+A of CD41-42 deletion mutantion (41.6%), IVS-2nt654(C → T) sudden change (21.8%), CD17(A → T) nonsense mutation (18.0%), TATA box nt-28(A → G)) phase shift mutation (3.9%), TATA box nt-29 suddenly change (1.2%); The gene frequency of these six kinds of sudden changes accounts for more than 94% of poor gene of Chinese ground, and wherein the CD41-42 deletion mutantion is the modal mutation types of Chinese.Therefore, the genes involved type is diagnosed accurately, be significant for β-thalassemia diagnosis with control.
Existing beta Thalassemia molecular diagnosis method commonly used comprises in the prior art: PCR extends amplification PCR (mutant oligonu2cleo tide extension amplification in conjunction with special oligonucleotide probe (PCR-ASO) spot hybridization, reverse hybridized (RDB) method, sudden change oligonucleotide, MOEA) technology, multiplex allele-specific PCR (multiplex allele specific PCR, MAS-PCR) method, allele specific PCR (allele specific, A SPCR), gene chips etc.Wherein, only for a kind of sudden change, the diagnosis of generally finishing unknown sample needs repeatedly duplicate detection to PCR usually, so rather waste time and energy in conjunction with special oligonucleotide probe (PCR-ASO) spot hybridization one-time detection.The reasons such as reverse hybridized (RDB) method exists pcr amplification bad, and the too weak or film bar washing of probe is insufficient on the Hybond membrane bar can cause the appearance of false positive or false negative result.The sudden change oligonucleotide extends the beta Thalassemia that the amplification round pcr is suitable for detecting the known dna point mutation, need carry out electrophoresis, wastes time and energy.Gene chips is easy, quick, micro-ization, automatization, once can the more even whole known mutations types of parallel examination novel method, detect but need buy expensive plant and instrument, be difficult to popularize.
Real-Time Fluorescent Quantitative PCR Technique (real-time fluorescent quantitative PCR, FQ-PCR) released by U.S. Applied Biosystems company in 1996, it is a kind of specific fluorescent probe that adds when adding pair of primers when pcr amplification, this probe is an oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the fluorescent signal of reporter group emission was absorbed by quenching group; When just beginning, probe is combined on the arbitrary strand of DNA; During pcr amplification, 5 ' end-3 ' the end 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal, it is DNA chain of every amplification, just there is a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form Complete Synchronization.This technology not only realized to dna profiling quantitatively, and have highly sensitive, specificity and the characteristics such as reliability is stronger, can realize multiple reaction, level of automation height, nonstaining property, tool real-time and accuracy, be widely used at present the fields such as molecular biology research and medical research.
Therefore, the plurality of advantages of combined with fluorescent quantitative PCR technology, developing a kind of can quick, accurate, the high sensitive fluorescent quantificationally PCR detecting kit that detects β-thalassemia gene specific locus mutation be the technical problem that industry needs to be resolved hurrily.
Summary of the invention
In order to overcome the defective of prior art, technical problem solved by the invention is to provide a kind of β-thalassemia sudden change fluorescent quantificationally PCR detecting kit, can be fast, accurately, the sudden change situation of high sensitive ground detection β-thalassemia gene, especially for 6 kinds of common β of Chinese-thalassemia gene specific locus mutation.
β of the present invention-thalassemia sudden change fluorescent quantificationally PCR detecting kit, comprise PCR mixed reaction solution, positive reference substance and for detection of the fluorescent probe of β-thalassemia mutator gene type, comprise the PCR primer of amplification mutational site constant gene segment C of living in the described PCR mixed reaction solution.
Preferably, described mutational site comprise the A of A corresponding to the C of the 654th correspondence of base deletion sudden change corresponding to beta-globin gene the 41/42nd amino acids, second intron of beta-globin gene → T base mutation, beta-globin gene the 17th amino acids → T base mutation, the 28th correspondence in beta-globin gene promoter upstream → G base mutation, beta-globin gene the 71/72nd amino acids corresponding+at least a in the G of the 5th correspondence of A base insertion mutation, beta-globin gene First Intron → C base mutation.
Wherein, described PCR primer comprises at least one group of following two groups of primer centerings:
For the primer in zone of living in, mutational site corresponding to beta-globin gene the 41/42nd amino acids pair, the sequence of its PCR forward primer is shown in SEQ ID NO:1, and the sequence of PCR reverse primer is shown in SEQ ID NO:2:
A1：5’-CATAACAGCATCAGGAGTGGACAG-3’(SEQ?ID?NO:1);
A2：5’-ACTGACTCTCTCTGCCTATTGGTCTATT-3’(SEQ?ID?NO:2);
For the primer in the zone of living in, mutational site of the 654th correspondence of second intron of beta-globin gene pair, the sequence of its PCR forward primer is shown in SEQ ID NO:5, and the sequence of PCR reverse primer is shown in SEQ ID NO:6:
B1：5’-TGGTAGCTGGATTGTAGCTGCTATTA-3’(SEQ?ID?NO:5);
B2：5’-TTGCACCATTCTAAAGAATAACAGTGA-3’(SEQ?ID?NO:6)；
For the primer in the zone of living in, mutational site of the 28th correspondence in beta-globin gene promoter upstream pair, the sequence of its PCR forward primer is shown in SEQ ID NO:9, and the sequence of PCR reverse primer is shown in SEQ ID NO:10:
C1：5’-CTCCTTAAACCTGTCTTGTAACCTTGATA-3’(SEQ?ID?NO:9)；
C2：5’-TGAGGAGAAGTCTGCCGTTACTG-3’(SEQ?ID?NO:10)；
For the primer in zone of living in, mutational site corresponding to beta-globin gene the 17th amino acids pair, the sequence of its PCR forward primer is shown in SEQ ID NO:13, and the sequence of PCR reverse primer is shown in SEQ ID NO:14:
D1：5’-GTGTCAGAAGCAAATGTAAGCAATAGAT-3’(SEQ?ID?NO:13)；
D2：5’-TGTCATCACTTAGACCTCACCCTGT-3’(SEQ?ID?NO:14)；
For the primer in zone of living in, mutational site corresponding to beta-globin gene the 71/72nd amino acids pair, the sequence of its PCR forward primer is shown in SEQ ID NO:17, and the sequence of PCR reverse primer is shown in SEQ ID NO:18:
E1：5’-GCCCTTGAGGTTGTCCAGGT-3’(SEQ?ID?NO:17)；
E2：5’-TATGGGCAACCCTAAGGTGAAG-3’(SEQ?ID?NO:18)；
For the primer in the zone of living in, mutational site of the 5th correspondence of beta-globin gene First Intron pair, the sequence of its PCR forward primer is shown in SEQ ID NO:21, and the sequence of PCR reverse primer is shown in SEQ ID NO:22:
F1：5’-TGTCTCCACATGCCCAGTTTC-3’(SEQ?ID?NO:21)；
F2：5’-CCTGAGGAGAAGTCTGCCGTTA-3’(SEQ?ID?NO:22)。
Preferably, described fluorescent probe comprises at least a in the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23 and SEQ ID NO:24:
A3:5 '-HEX-AGGACTCAAAGAACCT-MGB-3 ' (SEQ ID NO:3, wild-type);
A4:5 '-FAM-AAGGACTCAACCTCT-MGB-3 ' (SEQ ID NO:4, mutant);
B3:5 '-HEX-TGCTATTGCCTTAACC-MGB-3 ' (SEQ ID NO:7, wild-type);
B4:5 '-FAM-TTGCTATTACCTTAACCC-MGB-3 ' (SEQ ID NO:8, mutant);
C3:5 '-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3 ' (SEQ ID NO:11, wild-type);
C4:5 '-FAM-CCACGTTCACCTAGCCCCACAGG – TAMRA-3 ' (SEQ ID NO:12, mutant);
D3:5 '-HEX-TGCCCTGACTTTTATGCCCAGCC-TAMRA-3 ' (SEQ ID NO:15, wild-type);
D4:5 '-FAM-TGCCCTGACTTCTATGCCCAGCC – TAMRA-3 ' (SEQ ID NO:16, mutant);
E3:5 '-HEX-CAGGCCATCACTAAAGGCACCGAG-TAMRA-3 ' (SEQ ID NO:19, wild-type);
E4:5 '-FAM-CAGGCCATCACTTAAAGGCACCGA – TAMRA-3 ' (SEQ ID NO:20, mutant);
F3:5 '-HEX-CCTTGATACCAACCTG-MGB-3 ' (SEQ ID NO:23, wild-type);
F4:5 '-FAM-CCTTGATAGCAACCTG – MGB-3 ' (SEQ ID NO:24, mutant).
Preferably, described fluorescent probe comprises in the following probe combinations at least one group:
Detect probe combinations SEQ ID NO:3 and the SEQ ID NO:4 of the wild type gene of mutational site corresponding to beta-globin gene the 41/42nd amino acids and corresponding zone;
Probe combinations SEQ ID NO:7 and the SEQ ID NO:8 of the mutational site of the 654th correspondence of second intron of detection beta-globin gene and the wild type gene of corresponding zone;
Probe combinations SEQ ID SEQ ID NO:11 and the SEQ ID NO:12 of the mutational site of the 28th correspondence in detection beta-globin gene promoter upstream and the wild type gene of corresponding zone;
Detect probe combinations SEQ ID NO:15 and the SEQ ID NO:16 of the wild type gene of mutational site corresponding to beta-globin gene the 17th amino acids and corresponding zone;
Detect probe combinations SEQ ID NO:19 and the SEQ ID NO:20 of the wild type gene of mutational site corresponding to beta-globin gene the 71/72nd amino acids and corresponding zone;
Probe combinations SEQ ID NO:23 and the SEQ ID NO:24 of the mutational site of the 5th correspondence of detection beta-globin gene First Intron and the wild type gene of corresponding zone.
Preferably, described positive reference substance is the plasmid DNA that has beta-globin gene mutation, and the gene mutation site of beta-globin comprises the base deletion sudden change that beta-globin gene the 41/42nd amino acids is corresponding, the C of the 654th correspondence of second intron of beta-globin gene → T base mutation, A corresponding to beta-globin gene the 17th amino acids → T base mutation, the A of the 28th correspondence in beta-globin gene promoter upstream → G base mutation, beta-globin gene the 71/72nd amino acids is corresponding+A base insertion mutation, at least a in the G of the 5th correspondence of beta-globin gene First Intron → C base mutation.
Wherein, the wild-type probe A3:5 ' of the corresponding base of detection wild-type beta-globin gene the 41/42nd amino acids-HEX-AGGACTCAAAGAACCT-MGB-3 ' (SEQ ID NO:3), the probe A4:5 ' of the base deletion that detection beta-globin gene the 41/42nd amino acids is corresponding-FAM-AAGGACTCAACCTCT – MGB-3 ' (SEQ ID NO:4), therefore, when described fluorescent probe comprised such as one kind of SEQ ID NO:3 and SEQ ID NO:4 or two kinds, described positive reference substance was the plasmid DNA that has base deletion sudden change corresponding to beta-globin gene the 41/42nd amino acids.
In like manner, the wild-type probe B3:5 ' of the 654th corresponding base of second intron of detection wild-type beta-globin gene-HEX-TGCTATTGCCTTAACC-MGB-3 ' (SEQ ID NO:7), the probe B4:5 ' of the 654th bit base sudden change of second intron of detection beta-globin gene-FAM-TTGCTATTACCTTAACCC – MGB-3 ' (SEQ ID NO:8), when described fluorescent probe comprised such as one kind of SEQ ID NO:7 and SEQ ID NO:8 or two kinds, described positive reference substance was the plasmid DNA of the C that has the 654th correspondence of second intron of beta-globin gene → T base mutation.
The wild-type probe C3:5 ' of the corresponding base of detection wild-type beta-globin gene the 17th amino acids-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3 ' (SEQ ID NO:11), the probe C4:5 ' of the base mutation that detection beta-globin gene the 17th amino acids is corresponding-FAM-CCACGTTCACCTAGCCCCACAGG-TAMRA-3 ' (SEQ ID NO:12), when described fluorescent probe comprised such as one kind of SEQ ID NO:11 and SEQ ID NO:12 or two kinds, described positive reference substance was the plasmid DNA of the A that exists beta-globin gene the 17th amino acids corresponding → T base mutation.
The wild-type probe D3:5 ' of detection wild-type beta-globin gene promotor upstream the 28th bit base-HEX-TGCCCTGACTTTTATGCCCAGCC-TAMRA-3 ' (SEQ ID NO:15), the probe D4:5 ' of detection beta-globin gene promoter upstream the 28th bit base sudden change-FAM-TGCCCTGACTTCTATGCCCAGCC – TAMRA-3 ' (SEQ ID NO:16), when described fluorescent probe comprises such as one kind of SEQ ID NO:15 and SEQ ID NO:16 or two kinds, the plasmid DNA of the A of the 28th correspondence in beta-globin gene promoter upstream → G base mutation.
The wild-type probe E3:5 ' of the corresponding base of detection wild-type beta-globin gene the 71/72nd amino acids-HEX-CAGGCCATCACTAAAGGCACCGAG-TAMRA-3 ' (SEQ ID NO:19), the probe E4:5 ' of the base mutation that detection beta-globin gene the 71/72nd amino acids is corresponding-FAM-CAGGCCATCACTTAAAGGCACCGA – TAMRA-3 ' (SEQ ID NO:20), when described fluorescent probe comprises such as one kind of SEQ ID NO:19 and SEQ ID NO:20 or two kinds, beta-globin gene the 71/72nd amino acids is corresponding+plasmid DNA of A base insertion mutation.
The wild-type probe F3:5 ' of the 5th bit base of detection wild-type beta-globin gene First Intron-HEX-CCTTGATACCAACCTG-MGB-3 ' (SEQ ID NO:23), the probe F4:5 ' of the 5th bit base sudden change of detection beta-globin gene First Intron-FAM-CCTTGATAGCAACCTG – MGB-3 ' (SEQ ID NO:24), when described fluorescent probe comprises such as one kind of SEQ ID NO:23 and SEQ ID NO:24 or two kinds, the plasmid DNA of the G of the 5th correspondence of beta-globin gene First Intron → C base mutation.
Preferably, 5 ' end of described fluorescent probe is marked with the fluorescence report group, and 3 ' end of fluorescent probe is marked with the quenching of fluorescence group.Need to prove that above-mentioned probe sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23 and SEQ ID NO:24 are that corresponding nucleotides sequence is listed in specific implementation behind mark fluorescent reporter group and the fluorescent quenching group in the sequence table.In preferred implementation of the present invention, shown in above-mentioned probe sequence, 5 of (1) fluorescent probe SEQ ID NO:A3 ' end mark HEX fluorescence report group, 3 ' end mark MGB fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:A4 ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenching group.(2) 5 of fluorescent probe SEQ ID NO:B3 ' end mark HEX fluorescence report group, 3 ' end mark MGB fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:B4 ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenching group.(3) 5 of fluorescent probe SEQ ID NO:C3 ' end mark HEX fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:C4 ' end flag F AM fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group.(4) 5 of fluorescent probe SEQ ID NO:D3 ' end mark HEX fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:D4 ' end flag F AM fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group.(5) 5 of fluorescent probe SEQ ID NO:E3 ' end mark HEX fluorescence report group, 3 ' end is all remembered TAMRA fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:E4 ' end flag F AM fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group.(6) 5 of fluorescent probe SEQ ID NO:F3 ' end mark HEX fluorescence report group, 3 ' end mark MGB fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:F4 ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenching group.Certainly in of the present invention other are implemented; the different fluorescence report group of mark is just to distinguish for the fluorescence that quantitative fluorescent PCR can collect different wave length; therefore adopt the published fluorescence report group of other art technology and fluorescent quenching group; not affecting in the situation that detects effect, also should be understood to belong to protection scope of the present invention.
In principle, β-thalassemia sudden change fluorescent quantificationally PCR detecting kit is to adopt fluorescence quantifying PCR method that β-thalassemia transgenation is detected.For β-thalassemia gene mutation site respectively special design wild-type TaqMan probe and mutant TaqMan probe each is a pair of, when running into the wild-type nucleotide sequence, wild-type TaqMan probe can be with it fully combination, in the pcr amplification process, send corresponding fluorescent signal and be detected; And when running into the mutant nucleotide sequence, the with it fully combination of mutant TaqMan probe, the fluorescence report group sends fluorescence because enzymolysis separates, and can detect in real time the accumulation of corresponding fluorescent signal, thereby realizes detecting the purpose of β-thalassemia gene corresponding positions point mutation state.
Two ends provided by the invention all indicate the specificity fluorescent probe of fluorescence radiation group, when probe is complete, two groups distance on space structure is mutually close, 5 ' end fluorescence of sending of reporter group is because FRET (fluorescence resonance energy transfer) (FRET) and by the group cancellation of going out of 3 ' end quenching, so there is not the variation of fluorescent signal in the system.In case and it with sudden change after the combination of template specificity, its binding site is between two primers, extension along with primer, the Taq archaeal dna polymerase runs into the probe that combines with template in the chain extension process, its 5 '-3 ' 5 prime excision enzyme activity will cut off probe, the fluorescence report group has so just destroyed the FRET between two fluorophors away from the fluorescent quenching group, and the photofluorometer that the fluorescence that reporter group discharges just can be built in the instrument detects.PCR is every through a circulation, and fluorescent signal is also the same with the purpose fragment, and the process that has sync index to increase, the power of fluorescent signal have just represented what of copy number of template DNA.Therefore the present invention not only can be used for simple qualitative detection, also can be used as the detection by quantitative of the concrete content of sudden change sample.
Than existing detection method, the sudden change that the present invention adopts fluorescent quantitative PCR technique to detect β-thalassemia gene has following advantage:
1, detection sensitivity is high, and specificity is good: the present invention is directed to wild-type and mutant site and designed respectively two specificity fluorescent probes, improve detection sensitivity and specificity, false positive is low.
2, linear relationship is good, but detection by quantitative, because the power of fluorescent signal and the logarithm of template amplification product are linear, detection by fluorescent signal is carried out quantitatively sample original template concentration, error is little, other present detection methods can only be carried out qualitative detection, and FQ-PCR can realize real detection by quantitative.
3, present technique is lower to the specification of quality that specimen dna obtains, and no matter is paraffin organization or flesh tissue, can both obtain desirable detected result, and PCR-ASO, RDB method etc. are all higher to the requirement of sample, often must experience a plurality of loaded down with trivial details treatment steps.
4, lack detection time, can finish with interior at 12 hours to obtaining a result from specimen transfer; And, there is not aftertreatment, need not hybridization, electrophoresis, take pictures.
5, simple to operate, level of automation is high, and the FQ-PCR technology is to the amplification of PCR product and detect that next step is finished in the situation of stopped pipe, does not need to uncap, and crossed contamination and contaminate environment chance are few, have therefore also just reduced the probability of result error.
6, as a result interpretation is clear and definite, directly perceived; Also can carry out quantitative analysis to the result if need.Direct sequencing result's interpretation: need order-checking peak shape figure is manually read sequence, again the sequence of reading is analyzed together in conjunction with the original series in the gene pool, thereby the result of obtaining, fluorescence quantitative PCR method result's interpretation: more than the thresholding line, have the sample of amplification curve to be positive sample, as a result interpretation is very simple, and is directly perceived.
7, the sample size that detects is large, by high-throughput PCR instrument, once can detect 384 examples at most.
8, safety: do not comprise hazardous and noxious substances in the whole system, to operator and environment all without harm.
Description of drawings
Fig. 1 is the amplification curve diagram of base deletion sudden change (CD41-42 deletion mutantion) corresponding to fluorescence quantitative PCR detection beta-globin gene the 41/42nd amino acids;
Fig. 2 is the as a result figure that detects base deletion sudden change (CD41-42 deletion mutantion) corresponding to beta-globin gene the 41/42nd amino acids by direct sequencing;
Fig. 3 is the 654th bit base sudden change (IVS-II-654 (amplification curve diagram of C → T) of second intron of fluorescence quantitative PCR detection beta-globin gene;
Fig. 4 is the 654th bit base sudden change (IVS-II-654 (the as a result figure of C → T) that detects second intron of beta-globin gene by direct sequencing;
Fig. 5 is the amplification curve diagram of base mutation corresponding to fluorescence quantitative PCR detection beta-globin gene the 17th amino acids (CD17 (A → T));
Fig. 6 is the as a result figure that detects base mutation corresponding to beta-globin gene the 17th amino acids (CD17 (A → T)) by direct sequencing;
Fig. 7 is the amplification curve diagram of fluorescence quantitative PCR detection beta-globin gene promoter upstream the 28th bit base sudden change (TATA box nt-28 (A → G));
Fig. 8 is the as a result figure that detects beta-globin gene promoter upstream the 28th bit base sudden change (TATA box nt-28 (A → G)) by direct sequencing;
Fig. 9 is the amplification curve diagram of base mutation corresponding to fluorescence quantitative PCR detection beta-globin gene the 71/72nd amino acids (CD71-72 (+A));
Figure 10 is the as a result figure that detects base mutation corresponding to beta-globin gene the 71/72nd amino acids (CD71-72 (+A)) by direct sequencing;
Figure 11 is the amplification curve diagram of the 5th bit base sudden change (IVS-I-5 (G → C)) of fluorescence quantitative PCR detection beta-globin gene First Intron;
Figure 12 is the as a result figure that detects the 5th bit base sudden change (IVS-I-5 (G → C)) of beta-globin gene First Intron by direct sequencing;
Figure 13 is the amplification curve diagram of fluorescence quantitative PCR detection beta-globin gene mutation negative sample.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.
The sample explanation: used positive sample collection is the wax stone tissue of β-thalassemia specific locus mutation from Pathology Deparment of Tumor Hospital Attached to Zhongshan Univ. Clinicopathologic Diagnosis among the embodiment that gets off.From wax stone, extract genomic dna for following experimental applications.
Embodiment 1: the detection of the base deletion sudden change (CD41-42 deletion mutantion) that beta-globin gene the 41/42nd amino acids is corresponding
The sample explanation: it is the wax stone tissue of β-thalassemia specific locus mutation that positive sample is collected from Pathology Deparment of Tumor Hospital Attached to Zhongshan Univ. Clinicopathologic Diagnosis.From wax stone, extract genomic dna for following experimental applications.
Each is a pair of for the probe of the base deletion that design energy specific detection beta-globin gene the 41/42nd amino acids is corresponding and public primer:
Wherein, the sequence of PCR forward primer is shown in SEQ ID NO:1, and the sequence of PCR reverse primer is shown in SEQ ID NO:2:
A1：5’-CATAACAGCATCAGGAGTGGACAG-3’(SEQ?ID?NO:1);
A2：5’-ACTGACTCTCTCTGCCTATTGGTCTATT-3’(SEQ?ID?NO:2);
Fluorescent probe is:
A3：5’-HEX-AGGACTCAAAGAACCT-MGB-3’（SEQ?ID?NO:3）；
A4：5’-FAM-AAGGACTCAACCTCT-MGB-3’（SEQ?ID?NO:4）；
Wherein, the probe A3:5 ' of the corresponding base of detection wild-type beta-globin gene the 41/42nd amino acids-HEX-AGGACTCAAAGAACCT-MGB-3 ' (SEQ ID NO:3), the probe A4:5 ' of the base deletion that detection beta-globin gene the 41/42nd amino acids is corresponding-FAM-AAGGACTCAACCTCT – MGB-3 ' (SEQ ID NO:4), 5 of fluorescent probe SEQ ID NO:3 ' end mark HEX fluorescence report group, 3 ' end mark MGB fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:4 ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenching group.
Then optimizing reaction system is carried out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ M of positive and negative primer), each 0.2 μ l(20 μ M of probe), sample template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 64 ℃ 33 seconds, 45 circulations of amplified reaction.。
Simultaneously in the fluorescent quantitation test, when needing test sample, also need to arrange the sample reference material, validity with confirmed test, when described fluorescent probe comprised such as one kind of SEQ ID NO:3 and SEQ ID NO:4 or two kinds, described positive reference substance was the plasmid DNA that has base deletion sudden change corresponding to beta-globin gene the 41/42nd amino acids.
As shown in Figure 1, left side Grey curves is the corresponding hole fluorescent quantitative PCR curve that detects of CD41-42 mutant sample, the right black curve is the non-corresponding fluorescent quantitative PCR curve that detects the hole of CD41-42 mutant sample, namely normal and mutator gene all detects, so sample is the heterozygous mutant type.
In addition, the sample in above-described embodiment 1 is carried out sequence verification, find to detect the sample that test detects base deletion sudden change corresponding to beta-globin gene the 41/42nd amino acids by above-mentioned fluorescent quantitation in the present embodiment.As shown in Figure 2, can find out really by sequencing result in this site corresponding transgenation to occur that it is in full accord that itself and fluorescent quantitation detect the result of test.Find to adopt β of the present invention-thalassemia sudden change fluorescent quantificationally PCR detecting kit can determine exactly the base deletion sudden change situation that beta-globin gene the 41/42nd amino acids is corresponding with this.
Embodiment 2: the detection of the 654th bit base of second intron of beta-globin gene sudden change (IVS-II-654 (C → T))
Each is a pair of for the probe of the 654th bit base sudden change of second intron of design energy specific detection beta-globin gene and public primer:
Wherein, the sequence of PCR forward primer is shown in SEQ ID NO:5, and the sequence of PCR reverse primer is shown in SEQ ID NO:6:
B1：5’-TGGTAGCTGGATTGTAGCTGCTATTA-3’(SEQ?ID?NO:5);
B2：5’-TTGCACCATTCTAAAGAATAACAGTGA-3’(SEQ?ID?NO:6)；
Fluorescent probe is:
B3：5’-HEX-TGCTATTGCCTTAACC-MGB-3’（SEQ?ID?NO:7）；
B4：5’-FAM-TTGCTATTACCTTAACCC–MGB-3’（SEQ?ID?NO:8）；
Wherein, the probe B3:5 ' of the 654th corresponding base of second intron of detection wild-type beta-globin gene-HEX-TGCTATTGCCTTAACC-MGB-3 ' (SEQ ID NO:7), the probe B4:5 ' of the 654th bit base sudden change of second intron of detection beta-globin gene-FAM-TTGCTATTACCTTAACCC – MGB-3 ' (SEQ ID NO:8), 5 of fluorescent probe SEQ ID NO:7 ' end mark HEX fluorescence report group, 3 ' end mark MGB fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:8 ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenching group.
Then optimizing reaction system is carried out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ M of positive and negative primer), each 0.2 μ l(20 μ M of probe), sample template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 64 ℃ 33 seconds, 45 circulations of amplified reaction.。
Simultaneously in the fluorescent quantitation test, when needing test sample, also need to arrange the sample reference material, validity with confirmed test, when described fluorescent probe comprised such as one kind of SEQ ID NO:7 and SEQ ID NO:8 or two kinds, described positive reference substance was the plasmid DNA of the C that has the 654th correspondence of second intron of beta-globin gene → T base mutation.
As shown in Figure 3, the right Grey curves is that (the corresponding fluorescent quantitative PCR curve that detects the hole of the sudden change of C → T) sample, left side black curve are IVS-II-654 (the non-corresponding fluorescent quantitative PCR curve that detects the hole of sudden change sample of C → T) to IVS-II-654.
In addition, the sample in above-described embodiment 2 is carried out sequence verification, find to detect the sample that test detects the 654th bit base sudden change of second intron of beta-globin gene by above-mentioned fluorescent quantitation in the present embodiment.As shown in Figure 4, can find out really by sequencing result in this site corresponding transgenation to occur that it is in full accord that itself and fluorescent quantitation detect the result of test.Find to adopt β of the present invention-thalassemia sudden change fluorescent quantificationally PCR detecting kit can determine exactly the 654th bit base sudden change situation of second intron of beta-globin gene with this.
Embodiment 3: the detection of base mutation corresponding to beta-globin gene the 17th amino acids (CD17 (A → T))
Each is a pair of for the probe of the base mutation that design energy specific detection beta-globin gene the 17th amino acids is corresponding and public primer:
The sequence of PCR forward primer is shown in SEQ ID NO:9, and the sequence of PCR reverse primer is shown in SEQ ID NO:10:
C1：5’-CTCCTTAAACCTGTCTTGTAACCTTGATA-3’(SEQ?ID?NO:9)；
C2：5’-TGAGGAGAAGTCTGCCGTTACTG-3’(SEQ?ID?NO:10)；
Fluorescent probe is:
C3：5’-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3’（SEQ?ID?NO:11）；
C4：5’-FAM-CCACGTTCACCTAGCCCCACAGG-TAMRA-3’（SEQ?ID?NO:12）；
Wherein, the probe C3:5 ' of the corresponding base of detection wild-type beta-globin gene the 17th amino acids-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3 ' (SEQ ID NO:11), the probe C4:5 ' of the base mutation that detection beta-globin gene the 17th amino acids is corresponding-FAM-CCACGTTCACCTAGCCCCACAGG-TAMRA-3 ' (SEQ ID NO:12), 5 of fluorescent probe SEQ ID NO:11 ' end mark HEX fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:12 ' end flag F AM fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group.
Then optimizing reaction system is carried out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ M of positive and negative primer), each 0.2 μ l(20 μ M of probe), sample template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 64 ℃ 33 seconds, 45 circulations of amplified reaction.。
Simultaneously in the fluorescent quantitation test, when needing test sample, also need to arrange the sample reference material, validity with confirmed test, when described fluorescent probe comprised such as one kind of SEQ ID NO:11 and SEQ ID NO:12 or two kinds, described positive reference substance was the plasmid DNA of the A that exists beta-globin gene the 17th amino acids corresponding → T base mutation.
As shown in Figure 5, left side Grey curves is that (A → T) sudden change sample correspondence detects the fluorescent quantitative PCR curve in hole to CD17, and the right black curve is that (A → T) the non-correspondence of sudden change sample detects the fluorescent quantitative PCR curve in hole to CD17.
In addition, the sample in above-described embodiment 3 is carried out sequence verification, find to detect the sample that test detects base mutation corresponding to beta-globin gene the 17th amino acids by above-mentioned fluorescent quantitation in the present embodiment.As shown in Figure 6, can find out really by sequencing result in this site corresponding transgenation to occur that it is in full accord that itself and fluorescent quantitation detect the result of test.Find to adopt β of the present invention-thalassemia sudden change fluorescent quantificationally PCR detecting kit can determine exactly the base mutation situation that beta-globin gene the 17th amino acids is corresponding with this.
Embodiment 4: the detection of beta-globin gene promoter upstream the 28th bit base sudden change (TATA box nt-28 (A → G))
Each is a pair of for the probe of design energy specific detection beta-globin gene promoter upstream the 28th bit base sudden change and public primer:
Wherein, the sequence of PCR forward primer is shown in SEQ ID NO:13, and the sequence of PCR reverse primer is shown in SEQ ID NO:14:
D1：5’-GTGTCAGAAGCAAATGTAAGCAATAGAT-3’(SEQ?ID?NO:13)；
D2：5’-TGTCATCACTTAGACCTCACCCTGT-3’(SEQ?ID?NO:14)；
Fluorescent probe is:
D3：5’-HEX-TGCCCTGACTTTTATGCCCAGCC-TAMRA-3’（SEQ?ID?NO:15）；
D4：5’-FAM-TGCCCTGACTTCTATGCCCAGCC-TAMRA-3’（SEQ?ID?NO:16）；
The probe D3:5 ' of detection wild-type beta-globin gene promotor upstream the 28th bit base-HEX-TGCCCTGACTTTTATGCCCAGCC-TAMRA-3 ' (SEQ ID NO:15), the probe D4:5 ' of detection beta-globin gene promoter upstream the 28th bit base sudden change-FAM-TGCCCTGACTTCTATGCCCAGCC – TAMRA-3 ' (SEQ ID NO:16), 5 of fluorescent probe SEQ ID NO:15 ' end mark HEX fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:16 ' end flag F AM fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group.
Then optimizing reaction system is carried out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ M of positive and negative primer), each 0.2 μ l(20 μ M of probe), sample template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 64 ℃ 33 seconds, 45 circulations of amplified reaction.。
Simultaneously in the fluorescent quantitation test, when needing test sample, also need to arrange the sample reference material, validity with confirmed test, when described fluorescent probe comprises such as one kind of SEQ ID NO:15 and SEQ ID NO:16 or two kinds, the plasmid DNA of the A of the 28th correspondence in beta-globin gene promoter upstream → G base mutation.
As shown in Figure 7, left side Grey curves is that (A → G) sudden change sample correspondence detects hole fluorescent quantitative PCR curve to TATA box nt-28, and the right black curve is that (A → G) the non-correspondence of sudden change sample detects the fluorescent quantitative PCR curve in hole to TATA box nt-28.
In addition, the sample in above-described embodiment 4 is carried out sequence verification, find to detect the sample that test detects the 28th bit base sudden change of beta-globin gene promoter upstream by above-mentioned fluorescent quantitation in the present embodiment.As shown in Figure 8, can find out really by sequencing result in this site corresponding transgenation to occur that it is in full accord that itself and fluorescent quantitation detect the result of test.Find to adopt β of the present invention-thalassemia sudden change fluorescent quantificationally PCR detecting kit can determine exactly beta-globin gene promoter upstream the 28th bit base sudden change situation with this.
Embodiment 5: the detection of base mutation corresponding to beta-globin gene the 71/72nd amino acids (CD71-72 (+A))
Each is a pair of for the probe of the base mutation that design energy specific detection beta-globin gene the 71/72nd amino acids is corresponding and public primer:
Wherein, the sequence of PCR forward primer is shown in SEQ ID NO:17, and the sequence of PCR reverse primer is shown in SEQ ID NO:18:
E1：5’-GCCCTTGAGGTTGTCCAGGT-3’(SEQ?ID?NO:17)；
E2：5’-TATGGGCAACCCTAAGGTGAAG-3’(SEQ?ID?NO:18)；
Fluorescent probe is:
E3：5’-HEX-CAGGCCATCACTAAAGGCACCGAG-TAMRA-3’（SEQ?ID?NO:19）；
E4：5’-FAM-CAGGCCATCACTTAAAGGCACCGA–TAMRA-3’（SEQ?ID?NO:20）；
The probe E3:5 ' of the corresponding base of detection wild-type beta-globin gene the 71/72nd amino acids-HEX-CAGGCCATCACTAAAGGCACCGAG-TAMRA-3 ' (SEQ ID NO:19), the probe E4:5 ' of the base mutation that detection beta-globin gene the 71/72nd amino acids is corresponding-FAM-CAGGCCATCACTTAAAGGCACCGA-TAMRA-3 ' (SEQ ID NO:20), 5 of fluorescent probe SEQ ID NO:19 ' end mark HEX fluorescence report group, 3 ' end is all remembered TAMRA fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:20 ' end flag F AM fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group.
Then optimizing reaction system is carried out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ M of positive and negative primer), each 0.2 μ l(20 μ M of probe), sample template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 64 ℃ 33 seconds, 45 circulations of amplified reaction.
Simultaneously in the fluorescent quantitation test, when needing test sample, also need to arrange the sample reference material, validity with confirmed test, when described fluorescent probe comprises such as one kind of SEQ ID NO:19 and SEQ ID NO:20 or two kinds, beta-globin gene the 71/72nd amino acids is corresponding+plasmid DNA of A base insertion mutation.
As shown in Figure 9, the right blue curve is that (+A) the corresponding fluorescent quantitative PCR curve that detects the hole of sudden change sample, left side black curve are CD71-72 (+A) the non-corresponding fluorescent quantitative PCR curve that detects the hole of sudden change sample to CD71-72.
In addition, the sample in above-described embodiment 5 is carried out sequence verification, find to detect the sample that test detects base mutation corresponding to beta-globin gene the 71/72nd amino acids by above-mentioned fluorescent quantitation in the present embodiment.As shown in figure 10, can find out really by sequencing result in this site corresponding transgenation to occur that it is in full accord that itself and fluorescent quantitation detect the result of test.Find to adopt β of the present invention-thalassemia sudden change fluorescent quantificationally PCR detecting kit can determine exactly the base mutation situation that beta-globin gene the 71/72nd amino acids is corresponding with this.
Embodiment 6: the detection of the 5th bit base of beta-globin gene First Intron sudden change (IVS-I-5 (G → C))
Each is a pair of for the probe of the 5th bit base sudden change of design energy specific detection beta-globin gene First Intron and public primer:
Wherein, the sequence of PCR forward primer is shown in SEQ ID NO:21, and the sequence of PCR reverse primer is shown in SEQ ID NO:22:
F1：5’-TGTCTCCACATGCCCAGTTTC-3’(SEQ?ID?NO:21)；
F2：5’-CCTGAGGAGAAGTCTGCCGTTA-3’(SEQ?ID?NO:22)。
Fluorescent probe is:
F3：5’-HEX-CCTTGATACCAACCTG-MGB-3’（SEQ?ID?NO:23）；
F4：5’-FAM-CCTTGATAGCAACCTG-MGB-3’（SEQ?ID?NO:24）。
The probe F3:5 ' of the 5th bit base of detection wild-type beta-globin gene First Intron-HEX-CCTTGATACCAACCTG-MGB-3 ' (SEQ ID NO:23), the probe F4:5 ' of the 5th bit base sudden change of detection beta-globin gene First Intron-FAM-CCTTGATAGCAACCTG-MGB-3 ' (SEQ ID NO:24), 5 of fluorescent probe SEQ ID NO:23 ' end mark HEX fluorescence report group, 3 ' end mark MGB fluorescent quenching group; 5 of fluorescent probe SEQ ID NO:24 ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenching group.
Then optimizing reaction system is carried out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ M of positive and negative primer), each 0.2 μ l(20 μ M of probe), sample template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 64 ℃ 33 seconds, 45 circulations of amplified reaction.
Simultaneously in the fluorescent quantitation test, when needing test sample, also need to arrange the sample reference material, validity with confirmed test, when described fluorescent probe comprises such as one kind of SEQ ID NO:23 and SEQ ID NO:24 or two kinds, the plasmid DNA of the G of the 5th correspondence of beta-globin gene First Intron → C base mutation.
As shown in figure 11, left side Grey curves is that (G → C) sudden change sample correspondence detects the fluorescent quantitative PCR curve in hole to IVS-I-5, and the right black curve is that (G → C) the non-correspondence of sudden change sample detects the fluorescent quantitative PCR curve in hole to IVS-I-5.
In addition, the sample in above-described embodiment 6 is carried out sequence verification, find to detect the sample that test detects the 5th bit base sudden change of beta-globin gene First Intron by above-mentioned fluorescent quantitation in the present embodiment.As shown in figure 12, can find out really by sequencing result in this site corresponding transgenation to occur that it is in full accord that itself and fluorescent quantitation detect the result of test.Find to adopt β of the present invention-thalassemia sudden change fluorescent quantificationally PCR detecting kit can determine exactly the 5th bit base sudden change situation of beta-globin gene First Intron with this.
Embodiment 7: the detection of negative sample
With the reaction solution among the embodiment 1-6 negative sample is carried out fluorescence respectively, quantitative PCR detection, detected result as shown in Figure 7, blue six curves in the right are the corresponding fluorescent quantitative PCR curve that detects the hole of sample, and six black curves in the left side are the non-corresponding fluorescent quantitative PCR curve that detects the hole of sample.
Can find out from check result, do not detect in the sample CD41-42, IVS-II-654 (C → T), CD17 (A → T), TATA box nt-28 (A → G), CD71-72 (+A), (any in 6 kinds of mutator gene types of G → C), it is negative to be defined as β-thalassemia for IVS-I-5.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.
Claims (8)
1. a β-thalassemia sudden change fluorescent quantificationally PCR detecting kit, it is characterized in that, comprise PCR mixed reaction solution, positive reference substance and for detection of the fluorescent probe of β-thalassemia mutator gene type, comprise the PCR primer of amplification mutational site constant gene segment C of living in the described PCR mixed reaction solution.
2. β according to claim 1-thalassemia sudden change fluorescent quantificationally PCR detecting kit, it is characterized in that described mutational site comprises the base deletion sudden change that beta-globin gene the 41/42nd amino acids is corresponding, the C of the 654th correspondence of second intron of beta-globin gene → T base mutation, A corresponding to beta-globin gene the 17th amino acids → T base mutation, the A of the 28th correspondence in beta-globin gene promoter upstream → G base mutation, beta-globin gene the 71/72nd amino acids is corresponding+A base insertion mutation, at least a in the G of the 5th correspondence of beta-globin gene First Intron → C base mutation.
3. β according to claim 1-thalassemia sudden change fluorescent quantificationally PCR detecting kit is characterized in that described PCR primer comprises at least one group of following two groups of primer centerings:
For the primer in zone of living in, mutational site corresponding to beta-globin gene the 41/42nd amino acids pair, the sequence of its PCR forward primer is shown in SEQ ID NO:1, and the sequence of PCR reverse primer is shown in SEQ ID NO:2;
For the primer in the zone of living in, mutational site of the 654th correspondence of second intron of beta-globin gene pair, the sequence of its PCR forward primer is shown in SEQ ID NO:5, and the sequence of PCR reverse primer is shown in SEQ ID NO:6;
For the primer in the zone of living in, mutational site of the 28th correspondence in beta-globin gene promoter upstream pair, the sequence of its PCR forward primer is shown in SEQ ID NO:9, and the sequence of PCR reverse primer is shown in SEQ ID NO:10;
For the primer in zone of living in, mutational site corresponding to beta-globin gene the 17th amino acids pair, the sequence of its PCR forward primer is shown in SEQ ID NO:13, and the sequence of PCR reverse primer is shown in SEQ ID NO:14;
For the primer in zone of living in, mutational site corresponding to beta-globin gene the 71/72nd amino acids pair, the sequence of its PCR forward primer is shown in SEQ ID NO:17, and the sequence of PCR reverse primer is shown in SEQ ID NO:18;
For the primer in the zone of living in, mutational site of the 5th correspondence of beta-globin gene First Intron pair, the sequence of its PCR forward primer is shown in SEQ ID NO:21, and the sequence of PCR reverse primer is shown in SEQ ID NO:22.
4. β according to claim 1-thalassemia sudden change fluorescent quantificationally PCR detecting kit, it is characterized in that described fluorescent probe comprises at least a in the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23 and SEQ ID NO:24.
5. β according to claim 4-thalassemia sudden change fluorescent quantificationally PCR detecting kit is characterized in that described fluorescent probe comprises at least one group in the following probe combinations:
Detect probe combinations SEQ ID NO:3 and the SEQ ID NO:4 of the wild type gene of mutational site corresponding to beta-globin gene the 41/42nd amino acids and corresponding zone;
Probe combinations SEQ ID NO:7 and the SEQ ID NO:8 of the mutational site of the 654th correspondence of second intron of detection beta-globin gene and the wild type gene of corresponding zone;
Probe combinations SEQ ID SEQ ID NO:11 and the SEQ ID NO:12 of the mutational site of the 28th correspondence in detection beta-globin gene promoter upstream and the wild type gene of corresponding zone;
Detect probe combinations SEQ ID NO:15 and the SEQ ID NO:16 of the wild type gene of mutational site corresponding to beta-globin gene the 17th amino acids and corresponding zone;
Detect probe combinations SEQ ID NO:19 and the SEQ ID NO:20 of the wild type gene of mutational site corresponding to beta-globin gene the 71/72nd amino acids and corresponding zone;
Probe combinations SEQ ID NO:23 and the SEQ ID NO:24 of the mutational site of the 5th correspondence of detection beta-globin gene First Intron and the wild type gene of corresponding zone.
6. β according to claim 4-thalassemia sudden change fluorescent quantificationally PCR detecting kit, it is characterized in that, described positive reference substance is the plasmid DNA that has beta-globin gene mutation, and the gene mutation site of beta-globin comprises the base deletion sudden change that beta-globin gene the 41/42nd amino acids is corresponding, the C of the 654th correspondence of second intron of beta-globin gene → T base mutation, A corresponding to beta-globin gene the 17th amino acids → T base mutation, the A of the 28th correspondence in beta-globin gene promoter upstream → G base mutation, beta-globin gene the 71/72nd amino acids is corresponding+A base insertion mutation, at least a in the G of the 5th correspondence of beta-globin gene First Intron → C base mutation.
7. β according to claim 5-thalassemia sudden change fluorescent quantificationally PCR detecting kit is characterized in that:
When described fluorescent probe comprised such as one kind of SEQ ID NO:3 and SEQ ID NO:4 or two kinds, described positive reference substance was the plasmid DNA that has base deletion sudden change corresponding to beta-globin gene the 41/42nd amino acids;
When described fluorescent probe comprised such as one kind of SEQ ID NO:7 and SEQ ID NO:8 or two kinds, described positive reference substance was the plasmid DNA of the C that has the 654th correspondence of second intron of beta-globin gene → T base mutation;
When described fluorescent probe comprised such as one kind of SEQ ID NO:11 and SEQ ID NO:12 or two kinds, described positive reference substance was the plasmid DNA of the A that exists beta-globin gene the 17th amino acids corresponding → T base mutation;
When described fluorescent probe comprises such as one kind of SEQ ID NO:15 and SEQ ID NO:16 or two kinds, the plasmid DNA of the A of the 28th correspondence in beta-globin gene promoter upstream → G base mutation;
When described fluorescent probe comprises such as one kind of SEQ ID NO:19 and SEQ ID NO:20 or two kinds, beta-globin gene the 71/72nd amino acids is corresponding+plasmid DNA of A base insertion mutation;
When described fluorescent probe comprises such as one kind of SEQ ID NO:23 and SEQ ID NO:24 or two kinds, the plasmid DNA of the G of the 5th correspondence of beta-globin gene First Intron → C base mutation.
8. β according to claim 1-thalassemia sudden change fluorescent quantificationally PCR detecting kit is characterized in that, 5 ' end of described fluorescent probe is marked with the fluorescence report group, and 3 ' end of fluorescent probe is marked with the quenching of fluorescence group.
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