CN104141014A - Kit for rapidly detecting common deletion type alpha-thalassemia and use method of kit - Google Patents

Kit for rapidly detecting common deletion type alpha-thalassemia and use method of kit Download PDF

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CN104141014A
CN104141014A CN201410417598.4A CN201410417598A CN104141014A CN 104141014 A CN104141014 A CN 104141014A CN 201410417598 A CN201410417598 A CN 201410417598A CN 104141014 A CN104141014 A CN 104141014A
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龙驹
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Abstract

The invention discloses a kit for rapidly detecting common deletion type alpha-thalassemia and a use method of the kit. The kit comprises a pair of primers which can be used for simultaneously amplifying a characteristic sequence y1 in a section Y1 and a characteristic sequence y2 in a section Y2 in an alpha-globin gene cluster, a pair of primers for amplifying an --<SEA> gene type in the alpha-globin gene cluster, a pair of primers for amplifying alpha2 genes in the alpha-globin gene cluster, a fluorescent probe for specifically detecting the characteristic sequence y1 in the section Y1, a fluorescent probe for specifically detecting the characteristic sequence y2 in the section Y2, a fluorescent probe for specifically detecting --<SEA> gene type amplification products and a fluorescent probe for specifically detecting alpha2 gene amplification products. The kit disclosed by the invention has high sensitivity, stability and accuracy and relative high specificity in detection of alpha-thalassemia globin gene deletion.

Description

A kind of test kit and using method thereof of rapid detection common deletion type α-thalassemia
Technical field
The present invention relates to technical field of medical detection, be specifically related to a kind of test kit and using method thereof of rapid detection common deletion type α-thalassemia.
Background technology
Thalassemia (Mediterranean Anemia), also claims Thalassemia (Thalassemia), Ku Le (Cooley) anaemia, globin dyssynthesis anaemia, is called for short poor.Thalassemia is one of modal monogenic inheritance disease, and the poor kind in common ground has that α-ground is poor and β-ground is poor, is caused respectively by α-protein gene bunch and beta-globin gene cluster unconventionality expression.In China, Guangxi, Guangdong and Hainan are thalassemic provinces occurred frequently, and wherein the poor sickness rate in α-ground, Guangxi is 15.5%.In Chinese population, the poor common genotype of genotype in absence type α-ground is-- sEA,-α 3.7with-α 4.2.Detect at present the thalassemic main method of absence type and have Gap-PCR, high resolution solubility curve (HRM, High-Resolution Melting), MLPA (Multiplex Ligation-Dependent Probe Amplification Assay), Southern blot analytical method etc.Above method is applied in separately respectively relative merits in thalassemia genotype detection.
Homologous gene quantitative technique is a kind of according to the homology of gene, adopts common amplimer to increase to homologous gene, finally adopts fluorescent marker method or probe method the content of gene to be carried out to the method for relative quantification.α-globin gene cluster (NG_000006.1) is by regulatory factor (HS-40), α gene (α 1 and α 2), ζ gene (ζ 2), pseudogene (Ψ ζ 1, Ψ α 2and Ψ α 1) and θ composition, it puts in order as HS40 – ζ 2 – Ψ ζ 1 – Ψ α 2 – Ψ α 1 – α 2 – α 1 – θ.Gene of alpha thalassemia, according to its homology, can be divided into X1, X2, Y1, Y2, Z1 and Z2 interval, wherein-α 3.7the formation of (also referred to as rightward deletion) comes from the homologous recombination between Z1 and Z2, and-α 4.2(also referred to as lefrward deletion) derives from the homologous recombination between X1 and X2, as shown in Figure 1, in Fig. 1, Y1 and Y2 homology.Have not yet to see the relevant report of the test kit of the rapid detection common deletion type α-thalassemia of the combination Taqman technology being formed by the amplimer of increase Y1 and Y2 simultaneously.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of test kit and using method thereof of the rapid detection common deletion type α-thalassemia based on homologous gene quantitative technique.Adopt this test kit to detect α 1 and the copy number of α 2 functional genes and the variation of copy number in α-globin gene cluster by relative quantification.
The test kit of rapid detection common deletion type α-thalassemia of the present invention, comprise amplimer and fluorescent probe, described amplimer is: primer Y1Y2-F and the Y1Y2-R of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section in a pair of α-globin gene cluster that can simultaneously increase, in pair for amplification α-globin gene cluster-- sEAgenotypic Gap-PCR primer SEA-F and SEA-R, and primer A2-F and the A2-R of α 2 genes in pair for amplification α-globin gene cluster; Described fluorescent probe is: the fluorescent probe Y1-Prob of the characteristic sequence y1 of a specific detection Y1 section, and the fluorescent probe Y2-Prob of the characteristic sequence y2 of a specific detection Y2 section, a specific detection-- sEAthe fluorescent probe SEA-Prob of genotype amplified production, and the fluorescent probe A2-Prob of specific detection α 2 gene amplification products;
Wherein:
In the described α-globin gene cluster that can simultaneously increase in the primer pair of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section,
Y1Y2-F:5’-TGCACCCACTGGCACTC-3’；
Y1Y2-R:5’-AGGAAGGAAGGGGTGGACT-3’；
In described amplification α-globin gene cluster-- sEAin genotypic Gap-PCR primer pair,
SEA-F:5’-AGAAGCTGAGTGATGGGTCC-3’；
SEA-R:5’-TGGACTTAAGTGATCCTCCTGC-3’；
In described amplification α-globin gene cluster in the primer pair of α 2 genes,
A2-F:5’-TCCTGGCTTCTGTGAGCAC-3’；
A2-R:5’-GCAGAGAGGTCCTTGGTCTG-3’；
The fluorescent probe Y1-Prob of the characteristic sequence y1 of described specific detection Y1 section is:
Y1-Prob:5’-6-FAM-CACCTCCCACCCTTCCCCAGA-BHQ-1-3’；
The fluorescent probe Y2-Prob of the characteristic sequence y2 of described specific detection Y2 section is:
Y2-Prob:5’-ROX-CTCCCACCCTCCCCCTCGCCA-BHQ-2-3’；
Described specific detection-- sEAthe fluorescent probe SEA-Prob of genotype amplified production is:
SEA-Prob:5’-HEX-CTTCGCAGGAACTCGGTCGTCCC-BHQ-1-3’；
The fluorescent probe A2-Prob of described specific detection α 2 gene amplification products is:
A2-Prob:5’-CY5-ACCTCCATTGTTGGCACATTCCGGGA-BHQ-2-3’。
In fluorescent probe of the present invention, FAM refers to Fluoresceincarboxylic acid, and HEX refers to chlordene-6-methyl fluorescein, and ROX refers to carboxyl-X-rhodamine, and CY5 refers to cyanine dyes molecule 5, and BHQ-1 and BHQ-2 refer to fluorescent quenching group.
In technique scheme, the sequence of described Y1 section is: 5 '-AGTCCACCCCTTCCTTCCTCACCCTGCAGGAGCTGGCCAGCCTCATCACCCCAACA TCTCCCCACCTCCATTCTCCAACCACAGGGCCCTTGTCTCCTCTGTCCTTTCCCCT CCCCGAGCCAAGCCTCCTCCCTCCTCCACCTCCTCCACCTAATACATATCCTTAAG TCTCACCTCCTCCAGGAAGCCCTCAGACTAACCCTGGTCACCTTGAATGCCTCGTC CACACCTCCAGACTTCCTCAGGGCCTGTGATGAGGTCTGCACCTCTGTGTGTACTT GTGTGATGGTTAGAGGACTGCCTACCTCCCAGAGGAGGTTGAATGCTCCAGCCGGT TCCAGCTATTGCTTTGTTTACCTGTTTAACCAGTATTTACCTAGCAAGTCTTCCAT CAGATAG-3 '; The characteristic sequence y1 of this Y1 section is: 5 '-TGCACCCACTGGCACTCCTGCACCTCCCACCCTTCCCCAGAAGTCCACCCCTTCCT TCCT-3 '.
In technique scheme, the sequence of described Y2 section is: 5 '-AGTCCACCCCTTCCTTCCTCACCCCACATCCCCTCACCTACATTCTGCAACCACAG GGGCCTTCTCTCCCCTGTCCTTTCCCTACCCAGAGCCAAGTTTGTTTATCTGTTTA CAACCAGTATTTACCTAGCAAGTCTTCCATCAGATAG-3 '; The characteristic sequence y2 of this Y2 section is: 5 '-TGCACCCACTGGCACTCCTGCACCTCCCACCCTCCCCCTCGCCAAGTCCACCCCTT CCTTCCT-3 '.
The test kit of rapid detection common deletion type α-thalassemia of the present invention also comprises conventional in some available reagent boxes and necessary component, as damping fluid, enzyme liquid, MgCl 2with dNTP etc., concrete, enzyme liquid is archaeal dna polymerase, specifically can adopt the GoldStar Taq DNA Polymerase of health for producing in century, and damping fluid is the damping fluid supporting with GoldStar Taq DNA Polymerase.
The present invention also provides and adopts mentioned reagent box to carry out the method for rapid detection common deletion type α-thalassemia, comprises the following steps:
1) extracting sample genomic dna, prepares DNA profiling;
2) preparation reaction system, is specially:
Get in the primer Y1Y2-F of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section in the α-globin gene cluster that can simultaneously increase and Y1Y2-R, amplification α-globin gene cluster-- sEAprimer A2-F and the A2-R of α 2 genes in genotypic Gap-PCR primer SEA-F and SEA-R, amplification α-globin gene cluster; Fluorescent probe Y2-Prob, the specific detection of the fluorescent probe Y1-Prob of the characteristic sequence y1 of specific detection Y1 section, the characteristic sequence y2 of specific detection Y2 section-- sEAthe fluorescent probe A2-Prob of the fluorescent probe SEA-Prob of genotype amplified production and specific detection α 2 gene amplification products; And PCR damping fluid, enzyme liquid, MgCl 2, dNTP, water and DNA profiling be mixed with reaction system;
3) pattern detection: respectively the reaction system of preparation is carried out to pcr amplification, record the cycle index Cq that experiences when fluorescent signal in each PCR reaction tubes arrives the thresholding of setting (size of Cq value can reflect detected template number number);
4) data analysis and result are judged: the Cq value obtaining according to detection by quantitative, taking normal genotype (α α/α α) sample as contrast sample, calculate by following formula:
The gene Δ Cq=Cq of sample to be checked _ Y2-Cq _ Y1;
The gene Δ Δ Cq=Δ Cq of sample to be checked _ sample to be checked-Δ Cq _ normal sample;
The Y1 of sample to be checked and the relative copy number ratio of Y2 (using Y1 and Y2 here)
Carry out result judgement according to the value of R in conjunction with following table 1:
Table 1:
Genotype Y2/Y1 (theoretical value) SEA A2
αα/αα 1 - +
-- SEA/αα 1 + +
-α 3.7/αα 2 - +
-α 4.2/αα 0.5 - +
-- SEA/-α 3.7 Y2+，Y1- + -
-- SEA/-α 4.2 Y1+，Y2- + -
-α 3.7/-α 3.7 Y2+，Y1- - -
-α 4.2/-α 4.2 Y1+，Y2- - -
-α 3.7/-α 4.2 1 - -
-- SEA/-- SEA Y1-，Y2- + -
Wherein, when R is during in 0.75～1.26 scope, its result matches " 1 " in table theoretical value; When R is during in 1.8～2.6 scope, its result matches " 2 " in table theoretical value; When R is during in 0.33～0.53 scope, its result matches " 0.5 " in table theoretical value; If the calculation result of R not within the scope of R value defined above time, should adopt additive method to carry out auxiliary judgment; In table, "+" represents the positive, represents to exist amplified production; "-" represents negative, do not have this amplified production.
Ultimate principle of the present invention is to be the pathogenesis that causes the poor basic reason in α-ground based on α-globin genetically deficient.-α 3.7with-α 4.2the Molecular Biology Mechanism as shown in Figure 1.While adopting test kit of the present invention to detect in conjunction with aforesaid method, work as Y1=Y2, and A2 is while having amplification curve, do not comprise-α of sample to be tested 3.7or-α 4.2; If Y1=2Y2, and A2 is while having amplification curve, contain-α of the genotype of sample to be tested 4.2, without other α disappearances; If 2Y1=Y2, and A2 is while having amplification curve, contain-α of the genotype of sample to be tested 3.7, without other α disappearances; If Y1=0, must reference-- sEAif SEA has amplification curve, the genotype of this sample is-- sEAwith-α 3.7the genotype of composition, if SEA does not have amplification curve, this sample genotype is-α 3.7/-α 3.7; If Y2=0, must reference-- sEAif SEA has amplification curve, the genotype of this sample is-- sEAwith-α 4.2composition genotype, if without SEA amplification curve this sample genotype be-α 4.2/-α 4.2; If Y1=Y2 and A2 be without amplification curve, and SEA is during without amplification curve, and the poor genotype in the α-ground of sample to be tested is-α 3.7/-α 4.2; If while only having SEA to have amplification curve, this sample genotype is-- sEA/-- sEA.
Test kit of the present invention, is to adopt quadruple TaqMan fluorescent quantitation technology, and the copy number of α 1, α 2 genes in detection by quantitative sample, and the having or not of SEA and A2, comprehensively realize the poor rapid molecular diagnosis in common deletion type α-ground.Due to TaqMan fluorescent quantitation technology in use, its quantitative result is not to follow perfect theoretical value, its result can be floated in the left and right of theoretical value, therefore, the applicant's statistics after to the checking of a large amount of known type samples is just drawn the corresponding relation in theoretical value in the calculated value of the R described in aforesaid method and table 1, if the calculation result of R not within the scope of R value defined above time, should adopt additive method to carry out auxiliary judgment.
The step 1 of aforesaid method) in, adopt existing ordinary method to prepare DNA profiling.
The step 2 of aforesaid method) in:
In the described α-globin gene cluster that can simultaneously increase in the primer pair of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section,
Y1Y2-F:5’-TGCACCCACTGGCACTC-3’；
Y1Y2-R:5’-AGGAAGGAAGGGGTGGACT-3’；
In described amplification α-globin gene cluster-- sEAin genotypic Gap-PCR primer pair,
SEA-F:5’-AGAAGCTGAGTGATGGGTCC-3’；
SEA-R:5’-TGGACTTAAGTGATCCTCCTGC-3’；
In described amplification α-globin gene cluster in the primer pair of α 2 genes,
A2-F:5’-TCCTGGCTTCTGTGAGCAC-3’；
A2-R:5’-GCAGAGAGGTCCTTGGTCTG-3’；
The fluorescent probe Y1-Prob of the characteristic sequence y1 of described specific detection Y1 section is:
Y1-Prob:5’-6-FAM-CACCTCCCACCCTTCCCCAGA-BHQ-1-3’；
The fluorescent probe Y2-Prob of the characteristic sequence y2 of described specific detection Y2 section is:
Y2-Prob:5’-ROX-CTCCCACCCTCCCCCTCGCCA-BHQ-2-3’；
Described specific detection-- sEAthe fluorescent probe SEA-Prob of genotype amplified production is:
SEA-Prob:5’-HEX-CTTCGCAGGAACTCGGTCGTCCC-BHQ-1-3’；
The fluorescent probe A2-Prob of described specific detection α 2 gene amplification products is:
A2-Prob:5’-CY5-ACCTCCATTGTTGGCACATTCCGGGA-BHQ-2-3’；
In fluorescent probe, FAM refers to Fluoresceincarboxylic acid, and HEX refers to chlordene-6-methyl fluorescein, and ROX refers to carboxyl-X-rhodamine, and CY5 refers to cyanine dyes molecule 5, and BHQ-1 and BHQ-2 refer to fluorescent quenching group.
The step 2 of aforesaid method) in, in described reaction system, the concentration of each component is preferably: each primer: 0.25～0.5 μ mol/L; DNA profiling: 20～200ng; Magnesium ion: 1.5～1.9mmol/L; The final volume of reaction system is preferably 20 μ L.
The step 3 of aforesaid method) in, pcr amplification condition is: 95 DEG C of denaturations 8～10 minutes, then 95 DEG C 15～30 seconds, 60 DEG C of annealing 50～70 seconds, 39～49 circulations, gather fluorescent signals in 60 DEG C of annealing steps ends.Preferred pcr amplification condition is: 95 DEG C of denaturations 10 minutes, then 95 DEG C 15 seconds, 60 DEG C of annealing 60 seconds, 40 circulations.
Compared with prior art, feature of the present invention is:
1, in test kit of the present invention, need quantitative 2 genes (Y1 and Y2) only to adopt 1 group of primer can increase, ensured to greatest extent the consistence of amplification efficiency and quantitative accuracy;
2, test kit of the present invention, without introducing reference gene, adopts relative quantification method, by ratio result, and can accurately judge genotype in conjunction with having or not of SEA and A2 curve;
3, α-thalassemia globin gene disappearance is detected to susceptibility, stability and the accuracy with height, and higher specificity.
Brief description of the drawings
Fig. 1 is-α 3.7with-α 4.2molecular biology structure iron, from structural analysis in figure, the copy number ratio of Y1 and Y2 can be used as differentiation-α 3.7with-α 4.2foundation.
Embodiment
With specific embodiment, the invention will be further described below, but the present invention is not limited to these embodiment.
Embodiment 1: adopt the detected result of test kit of the present invention in known type sample
1, the composition of test kit:
(1) can simultaneously increase primer pair Y1Y2-F and the Y1Y2-R of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section in α-globin gene cluster:
Y1Y2-F:5’-TGCACCCACTGGCACTC-3’(SEQ?ID?NO：1)；
Y1Y2-R:5’-AGGAAGGAAGGGGTGGACT-3’(SEQ?ID?NO：2)；
(2) in amplification α-globin gene cluster-- sEAgenotypic Gap-PCR primer pair SEA-F and SEA-R:
SEA-F:5’-AGAAGCTGAGTGATGGGTCC-3’(SEQ?ID?NO：3)；
SEA-R:5’-TGGACTTAAGTGATCCTCCTGC-3’(SEQ?ID?NO：4)；
(3) primer pair A2-F and the A2-R of α 2 genes in amplification α-globin gene cluster:
A2-F:5’-TCCTGGCTTCTGTGAGCAC-3’(SEQ?ID?NO：5)；
A2-R:5’-GCAGAGAGGTCCTTGGTCTG-3’(SEQ?ID?NO：6)；
(4) the fluorescent probe Y1-Prob of the characteristic sequence y1 of specific detection Y1 section:
Y1-Prob:5’-6-FAM-CACCTCCCACCCTTCCCCAGA-BHQ-1-3’(SEQ?ID?NO：7)；
(5) the fluorescent probe Y2-Prob of the characteristic sequence y2 of specific detection Y2 section:
Y2-Prob:5’-ROX-CTCCCACCCTCCCCCTCGCCA-BHQ-2-3’(SEQ?ID?NO：8)；
(6) specific detection-- sEAthe fluorescent probe SEA-Prob of genotype amplified production:
SEA-Prob:5’-HEX-CTTCGCAGGAACTCGGTCGTCCC-BHQ-1-3’(SEQ?ID?NO：9)；
(7) the fluorescent probe A2-Prob of specific detection α 2 gene amplification products:
A2-Prob:5’-CY5-ACCTCCATTGTTGGCACATTCCGGGA-BHQ-2-3’(SEQ?ID?NO：10)；
Above-mentioned each primer and fluorescent probe are all for designing in α-globin gene cluster, wherein:
The sequence of described Y1 section is:
5 '-AGTCCACCCCTTCCTTCCTCACCCTGCAGGAGCTGGCCAGCCTCATCACCCCAACA TCTCCCCACCTCCATTCTCCAACCACAGGGCCCTTGTCTCCTCTGTCCTTTCCCCT CCCCGAGCCAAGCCTCCTCCCTCCTCCACCTCCTCCACCTAATACATATCCTTAAG TCTCACCTCCTCCAGGAAGCCCTCAGACTAACCCTGGTCACCTTGAATGCCTCGTC CACACCTCCAGACTTCCTCAGGGCCTGTGATGAGGTCTGCACCTCTGTGTGTACTT GTGTGATGGTTAGAGGACTGCCTACCTCCCAGAGGAGGTTGAATGCTCCAGCCGGT TCCAGCTATTGCTTTGTTTACCTGTTTAACCAGTATTTACCTAGCAAGTCTTCCAT CAGATAG-3 ' (SEQ ID NO:11); The characteristic sequence y1 of this Y1 section is: 5 '-TGCACCCACTGGCACTCCTGCACCTCCCACCCTTCCCCAGAAGTCCACCCCTTCCT TCCT-3 ' (SEQ ID NO:12);
The sequence of described Y2 section is:
5 '-AGTCCACCCCTTCCTTCCTCACCCCACATCCCCTCACCTACATTCTGCAACCACAG GGGCCTTCTCTCCCCTGTCCTTTCCCTACCCAGAGCCAAGTTTGTTTATCTGTTTA CAACCAGTATTTACCTAGCAAGTCTTCCATCAGATAG-3 ' (SEQ ID NO:13); The characteristic sequence y2 of this Y2 section is: 5 '-TGCACCCACTGGCACTCCTGCACCTCCCACCCTCCCCCTCGCCAAGTCCACCCCTT CCTTCCT-3 ' (SEQ ID NO:14).
(8) other moiety:
GoldStar Taq DNA Polymerase, with supporting damping fluid and the dNTPs of GoldStar Taq DNA Polymerase be all century purchased from health, MgCl 2purchased from Life Technology.
Prepare PCR reaction system by following table 2:
Table 2:(mM represents mmol/L, and μ M represents μ mol/L)
2, implementation method:
PCR reaction instrument is the real-time thermal cycler CFX96 of Bio-Rad.PCR response procedures is:
95 DEG C of denaturation 10min; 95 DEG C of 15sec+60 DEG C of 1min, 40 circulations, gather fluorescent signal in 60 DEG C of annealing steps ends.
Sample process: adopt Lab-Aid DNA mini extraction kid (Bio-V, Xiamen) test kit extracting DNA, be diluted to 20～200ng/ μ L with distilled water for subsequent use.DNA sample also can adopt other conventional DNA extraction methods to extract.
Pattern detection: by sample to be checked, and 3 parts of normal genotypes (α α/α α) sample, according to above-mentioned reaction system and response procedures, on quantitative real time PCR Instrument, carry out augmentation detection, record Cq value.
3, sample source: all sample standard deviations source conventional Gap-PCR technology of hanging oneself is determined the DNA sample of caryogram.
4, data analysis and result are judged: by formula of the present invention (Δ Cq=Cq _ Y2-Cq _ Y1) calculate the gene Δ Cq of normal sample gene Δ Cq and sample to be checked _ sample to be checked; Using the average delta Cq value of above-mentioned 3 parts of normal genotype samples as Δ Cq _ normal sample, result as described in Table 3, according to formula Δ Δ Cq=Δ Cq _ treat sample originally-Δ Cq _ normal samplecalculate Δ Δ Cq value, pass through formula calculate the ratio of Y1 and Y2, and by its result Y1 that definite detection obtains according to aforementioned table 1 judging criterion and the relative copy number of Y2, result is as shown in following table 3 and table 4:
Table 3:
Note: in table, "+" represents that this passage has amplification curve positive, and "-" represents that this passage is without amplification curve, negative.
Table 4:
Note: in table, "+" represents that this passage has amplification curve positive, and "-" represents that this passage is without amplification curve, negative.
From result, the present invention can distinguish by data analysis-- sEA/ α α genotype, α α/α α genotype ,-α 3.7/ α α genotype and-α 4.2/ α α genotype; And the genotype of all samples that detect is identical through the definite genotype of conventional Gap-PCR technology with it, illustrates that test kit of the present invention has good accuracy in the time of the detection to known type sample.
Embodiment 2: the detection effect of the present invention in random gene type sample.
1, the composition of test kit:
With embodiment 1.
2, implementation method:
With embodiment 1.
3, sample source:
Sample is 36 routine random samples (Xin Yue Bioisystech Co., Ltd provides by Jinan), is diluted to 20～200ng, as sample to be checked with distilled water), and the normal samples of 3 example of verifying through Gap-PCR.
4, data analysis and result are judged:
Calculate according to data analysis formula of the present invention, and by its result Y1 that definite detection obtains according to aforementioned table 1 judging criterion and the relative copy number of Y2, result is as shown in following table 5 and table 6; In above-mentioned sample being detected by test kit of the present invention and method, adopt existing Gap-PCR method to verify, result is as shown in following table 5 and table 6:
Table 5:
Note: in table, "+" represents that this passage has amplification curve positive, and "-" represents that this passage is without amplification curve, negative.
Table 6:
Note: in table, "+" represents that this passage has amplification curve positive, and "-" represents that this passage is without amplification curve, negative.
From the above results, adopt test kit of the present invention in conjunction with method of the present invention common deletion type α-thalassemia detect in, its result is consistent with Gap-PCR method.

Claims (7)

1. a test kit for rapid detection common deletion type α-thalassemia, comprises amplimer and fluorescent probe, it is characterized in that:
Described amplimer is: primer Y1Y2-F and the Y1Y2-R of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section in a pair of α-globin gene cluster that can simultaneously increase, in pair for amplification α-globin gene cluster-- sEAgenotypic Gap-PCR primer SEA-F and SEA-R, and primer A2-F and the A2-R of α 2 genes in pair for amplification α-globin gene cluster;
Described fluorescent probe is: the fluorescent probe Y1-Prob of the characteristic sequence y1 of a specific detection Y1 section, and the fluorescent probe Y2-Prob of the characteristic sequence y2 of a specific detection Y2 section, a specific detection-- sEAthe fluorescent probe SEA-Prob of genotype amplified production, and the fluorescent probe A2-Prob of specific detection α 2 gene amplification products;
Wherein:
In the described α-globin gene cluster that can simultaneously increase in the primer pair of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section,
Y1Y2-F:5’-TGCACCCACTGGCACTC-3’；
Y1Y2-R:5’-AGGAAGGAAGGGGTGGACT-3’；
In described amplification α-globin gene cluster-- sEAin genotypic Gap-PCR primer pair,
SEA-F:5’-AGAAGCTGAGTGATGGGTCC-3’；
SEA-R:5’-TGGACTTAAGTGATCCTCCTGC-3’；
In described amplification α-globin gene cluster in the primer pair of α 2 genes,
A2-F:5’-TCCTGGCTTCTGTGAGCAC-3’；
A2-R:5’-GCAGAGAGGTCCTTGGTCTG-3’；
The fluorescent probe Y1-Prob of the characteristic sequence y1 of described specific detection Y1 section is:
Y1-Prob:5’-6-FAM-CACCTCCCACCCTTCCCCAGA-BHQ-1-3’；
The fluorescent probe Y2-Prob of the characteristic sequence y2 of described specific detection Y2 section is:
Y2-Prob:5’-ROX-CTCCCACCCTCCCCCTCGCCA-BHQ-2-3’；
Described specific detection-- sEAthe fluorescent probe SEA-Prob of genotype amplified production is:
SEA-Prob:5’-HEX-CTTCGCAGGAACTCGGTCGTCCC-BHQ-1-3’；
The fluorescent probe A2-Prob of described specific detection α 2 gene amplification products is:
A2-Prob:5’-CY5-ACCTCCATTGTTGGCACATTCCGGGA-BHQ-2-3’。
2. the test kit of rapid detection common deletion type α-thalassemia according to claim 1, is characterized in that:
The sequence of described Y1 section is:
5’-AGTCCACCCCTTCCTTCCTCACCCTGCAGGAGCTGGCCAGCCTCATCACCCCAACATCTCCCCACCTCCATTCTCCAACCACAGGGCCCTTGTCTCCTCTGTCCTTTCCCCTCCCCGAGCCAAGCCTCCTCCCTCCTCCACCTCCTCCACCTAATACATATCCTTAAGTCTCACCTCCTCCAGGAAGCCCTCAGACTAACCCTGGTCACCTTGAATGCCTCGTCCACACCTCCAGACTTCCTCAGGGCCTGTGATGAGGTCTGCACCTCTGTGTGTACTTGTGTGATGGTTAGAGGACTGCCTACCTCCCAGAGGAGGTTGAATGCTCCAGCCGGTTCCAGCTATTGCTTTGTTTACCTGTTTAACCAGTATTTACCTAGCAAGTCTTCCATCAGATAG-3’；
The sequence of described Y2 section is:
5’-AGTCCACCCCTTCCTTCCTCACCCCACATCCCCTCACCTACATTCTGCAACCACAGGGGCCTTCTCTCCCCTGTCCTTTCCCTACCCAGAGCCAAGTTTGTTTATCTGTTTACAACCAGTATTTACCTAGCAAGTCTTCCATCAGATAG-3’。
3. the test kit of rapid detection common deletion type α-thalassemia according to claim 1, is characterized in that:
The characteristic sequence y1 of described Y1 section is: 5 '-TGCACCCACTGGCACTCCTGCACCTCCCACCCTTCCCCAGAAGTCCACCCCTTCCT TCCT-3 ';
The characteristic sequence y2 of described Y2 section is: 5 '-TGCACCCACTGGCACTCCTGCACCTCCCACCCTCCCCCTCGCCAAGTCCACCCCTT CCTTCCT-3 '.
4. the test kit of rapid detection common deletion type α-thalassemia according to claim 1, is characterized in that: described test kit also comprises PCR damping fluid, enzyme liquid, MgCl 2and dNTP.
5. adopt test kit described in claim 1 to carry out the method for rapid detection common deletion type α-thalassemia, comprise the following steps:
1) extracting sample genomic dna, prepares DNA profiling;
2) preparation reaction system, is specially:
Get in the primer Y1Y2-F of the characteristic sequence y1 of Y1 section and the characteristic sequence y2 of Y2 section in the α-globin gene cluster that can simultaneously increase and Y1Y2-R, amplification α-globin gene cluster-- sEAprimer A2-F and the A2-R of α 2 genes in genotypic Gap-PCR primer SEA-F and SEA-R, amplification α-globin gene cluster; Fluorescent probe Y2-Prob, the specific detection of the fluorescent probe Y1-Prob of the characteristic sequence y1 of specific detection Y1 section, the characteristic sequence y2 of specific detection Y2 section-- sEAthe fluorescent probe A2-Prob of the fluorescent probe SEA-Prob of genotype amplified production and specific detection α 2 gene amplification products; And PCR damping fluid, enzyme liquid, MgCl 2, dNTP, water and DNA profiling be mixed with reaction system;
3) pattern detection: respectively the reaction system of preparation is carried out to pcr amplification, the cycle index Cq experiencing while recording the thresholding of the fluorescent signal arrival setting in each PCR reaction tubes;
4) data analysis and result are judged: the Cq value obtaining according to detection by quantitative, taking normal genotype sample as contrast sample, calculate by following formula:
The gene Δ Cq=Cq of sample to be checked _ Y2-Cq _ Y1;
The gene Δ Δ Cq=Δ Cq of sample to be checked _ sample to be checked-Δ Cq _ normal sample;
The Y1 of sample to be checked copy number ratio relative to Y2
According to the value of R in conjunction with carrying out result judgement in following table 1:
Table 1:
Genotype Y2/Y1 (theoretical value) SEA A2 αα/αα 1 - + -- SEA/αα 1 + + -α 3.7/αα 2 - + -α 4.2/αα 0.5 - + -- SEA/-α 3.7 Y2+，Y1- + - -- SEA/-α 4.2 Y1+，Y2- + - -α 3.7/-α 3.7 Y2+，Y1- - - -α 4.2/-α 4.2 Y1+，Y2- - - -α 3.7/-α 4.2 1 - - -- SEA/-- SEA Y1-，Y2- + -
Wherein, when R is during in 0.75～1.26 scope, its result matches " 1 " in table theoretical value; When R is during in 1.8～2.6 scope, its result matches " 2 " in table theoretical value; When R is during in 0.33～0.53 scope, its result matches " 0.5 " in table theoretical value; If the calculation result of R not within the scope of R value defined above time, should adopt additive method to carry out auxiliary judgment.
6. method according to claim 5, is characterized in that: step 3) in, pcr amplification condition is: 95 DEG C of denaturations 8～10 minutes, then 95 DEG C 15～30 seconds, 60 DEG C of annealing 50～70 seconds, 39～49 circulations.
7. method according to claim 5, is characterized in that: pcr amplification condition is: 95 DEG C of denaturations 10 minutes, then 95 DEG C 15 seconds, 60 DEG C of annealing 60 seconds, 40 circulations.
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CN109112205A (en) * 2017-06-23 2019-01-01 陈治中 The primer sets and kit of 3 kinds of deletional α-thalassemias are detected for Genotyping
CN109112208A (en) * 2017-06-23 2019-01-01 陈治中 For detecting the primer sets and kit of deletion form α-thalassemia
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CN110438219A (en) * 2019-08-12 2019-11-12 广东省妇幼保健院 Primer, probe, kit and method based on the noninvasive pre-natal diagnosis Pasteur oedema tire of droplet type digital pcr
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