CN101413032A - Reagent kit for detecting deletion and mutation of alpha-globin gene - Google Patents
Reagent kit for detecting deletion and mutation of alpha-globin gene Download PDFInfo
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Abstract
The invention discloses a kit for detecting delection and mutation of an alfa-globin gene. The kit comprises the following group of primers: a primer 1 is combined with a second intron of an alfa 2 gene of an alfa-globin gene cluster close to a sequence of a third exon where 7 nucleotide pairs are lost compared with an alfa 1; the primer 2 is combined with a down stream region of a termination codon 3' of the alfa 2 gene of the alfa-globin gene cluster; and the primer 3 and the primer 4 are combined with genome DNA in up and down streams of the Southeast Asian delection region of the alfa-globin gene cluster respectively. The kit can be used to type a alfa-globin encoding gene carried by a sample , detecting carriers of defective genes before marriage and childbirth, and conducting prenatal diagnosis of a fetus after pregnancy so as to effectively prevent diseases from being passed on to a future generation and contributing to the improvement of population quality. Detection with the kid has the advantages of high speed, automation, high flux, no use of polluting medicines and the like.
Description
Technical field
The present invention relates to a kind of test kit that detects α-Zhu Danbai genetically deficient and sudden change.
Background technology
Thalassemia (Thalassemia) was at first described by Cooley and Lee in nineteen twenty-five, was found in the mediterranean region the earliest, was called thalassemia at that time, also claimed Thalassemia abroad.In fact, this disease spreads all over all over the world, with Mediterranean Zone, middle Africa, South East Asia, SOUTHERN CHINA is more sees that Black American's sickness rate is also higher.In China, Guangdong, Guangxi, Guizhou, Sichuan are multiple district.Thalassemia is a class because the autosomal inheritance defective causes the globin chain dyssynthesis, and make one or more globin quantity not sufficients or lack fully, thereby the easy dissolved destructive hemolytic anemia of red corpuscle.China natural science noun validation board suggestion title that this is sick changes thalassemia into, still is referred to as thalassemia traditionally, and it is poor to be called for short ground.
Thalassemia is because due to the disappearance or point mutation of globin gene.Globin in the normal adult oxyphorase mainly is made up of two kinds of peptide chains, i.e. α chain and β chain, and by its corresponding genes encoding, the disappearance of these genes or point mutation can cause the dyssynthesis of corresponding peptide chain respectively, cause the component of oxyphorase to change.α and beta Thalassemia are the most common.
Human a globin gene cluster is positioned at 16Pter-p13.3.Every karyomit(e) has 2 a globin genes that function is arranged, and dyad has 4 a globin genes.Most of a thalassemias (it is poor to be called for short a ground) are because due to the disappearance of a globin gene, minority is caused by point mutation.
The poor diagnostic method in α-ground is divided into two classes, and class methods are traditional diagnostic methods, as Hb electrophoresis, isoelectrofocusing, efficient liquid phase chromatographic analysis etc.; Another kind of method is exactly genetic analysis.Continuous development along with the DNA analysis technology, diagnose this patient and Disease-causing gene carrier's level to improve constantly, can detect the dcc gene carrier before marriage and childbirth, antenatal diagnosis is done to fetus in conceived back, effectively stop this type of disease to pass to the next generation, significant to improving the health of the people.
Summary of the invention
The purpose of this invention is to provide a kind of test kit that detects α-Zhu Danbai genetically deficient and sudden change.
The test kit of detection α-Zhu Danbai genetically deficient provided by the invention and sudden change is to produce amplified production through PCR, draw the test kit of α-Zhu Danbai genetically deficient and results of mutation then by solubility curve analysis and/or sex change high performance liquid chromatography, comprise following primer:
Disappearance zone, South East Asia is about the section of 20kb for 5 ' upstream from the Ψ α 2 of α-Zhu Danbai gene cluster until Θ gene 3 ' end.
As first group of primer, test kit provided by the invention also can comprise following second group of primer and the 3rd group of primer with primer 1, primer 2, primer 3 and primer 4:
Second group of primer is made up of primer 5 and primer 6, and primer 5 and primer 6 are incorporated into α-Zhu Danbai gene cluster-α respectively
3.7The genomic dna of the upstream and downstream in disappearance zone;
The 3rd group of primer is made up of primer 7 and primer 8, and primer 7 and primer 8 are incorporated into α-Zhu Danbai gene cluster-α respectively
4.2The genomic dna of the upstream and downstream in disappearance zone.
-α
3.7The disappearance zone is the fragment from the about 3.7kb between 2 to α 1 two genes of α.
-α
4.2Disappearance zone is the X2 homologous sequence box held from 5 of the Ψ α 1 ' fragment to 4.2kb the X1 homologous sequence box of α 1 gene 3 ' end.
Point mutation is refered in particular in described sudden change, the allelotrope α of sudden change
Tα represents, α
Tα comprises three types: α
CSα (Hb Constant Spring), α
QSα (Guangxi sudden change) and α
WSα.
The sequence of described primer 1 is shown in the sequence 1 of sequence table, the sequence of described primer 2 is shown in the sequence 2 of sequence table, the sequence of described primer 3 is shown in the sequence 3 of sequence table, the sequence of described primer 4 is shown in the sequence 4 of sequence table, the sequence of described primer 5 is shown in the sequence 5 of sequence table, the sequence of described primer 6 is shown in the sequence 6 of sequence table, and the sequence of described primer 7 is shown in the sequence 7 of sequence table, and the sequence of described primer 8 is shown in the sequence 8 of sequence table.
Described test kit also comprises nonspecific embedding fluorescence dye.
Described fluorescence dye is SYBR Green I.
Described test kit also comprises real-time fluorescence quantitative PCR instrument or sex change high performance liquid chromatograph.
Described test kit also comprises the conventional reagent of Realtime-PCR.
The conventional reagent of described Realtime-PCR comprises: PCR damping fluid, dNTPs, MgCl
2With the Taq archaeal dna polymerase.
Described test kit can be applicable to crowd's examination, gene diagnosis and the prenatal gene diagnosis of α-Di Zhonghaipinxue.
The present invention also protects the application of primer sets in preparing the test kit that detects α-Zhu Danbai genetically deficient and sudden change by solubility curve analysis and/or sex change high performance liquid chromatography that comprises primer 1, primer 2, primer 3 and primer 4;
Described primer 1 is incorporated into second intron of α-Zhu Danbai gene cluster α 2 genes and compares the sequence place that lacks 7 nucleotide pairs near the 3rd exon with α 1; Described primer 2 is incorporated into α-Zhu Danbai gene cluster α 2 gene terminator codons 3 ' catchment;
Described primer 3 and primer 4 are incorporated into the genomic dna of the upstream and downstream in disappearance zone, α-Zhu Danbai gene cluster South East Asia respectively.
Described primer sets also comprises primer 5, primer 6, primer 7 and primer 8;
Described primer 5 and primer 6 are incorporated into α-Zhu Danbai gene cluster-α respectively
3.7The genomic dna of the upstream and downstream in disappearance zone;
Described primer 7 and primer 8 are incorporated into α-Zhu Danbai gene cluster-α respectively
4.2The genomic dna of the upstream and downstream in disappearance zone.
The sequence of described primer 1 is shown in the sequence 1 of sequence table, the sequence of described primer 2 is shown in the sequence 2 of sequence table, the sequence of described primer 3 is shown in the sequence 3 of sequence table, the sequence of described primer 4 is shown in the sequence 4 of sequence table, the sequence of described primer 5 is shown in the sequence 5 of sequence table, the sequence of described primer 6 is shown in the sequence 6 of sequence table, and the sequence of described primer 7 is shown in the sequence 7 of sequence table, and the sequence of described primer 8 is shown in the sequence 8 of sequence table.
The application method and the interpretation method of test kit provided by the invention are as follows:
Extracting the genomic dna of sample, is template with the genomic dna, carries out the Realtime-PCR reaction of following three individual system respectively:
System first: carry out the PCR reaction with primer 1, primer 2, primer 3 and primer 4.
System second: carry out the PCR reaction with primer 5 and primer 6.
System third: carry out the PCR reaction with primer 7 and primer 8.
The PCR product is carried out the solubility curve analysis, and used fluorescence dye is SYBR Green I, and instrument slowly is incremented to 95 ℃ from 60 ℃ automatically, carries out interpretation as a result and makes diagnosis (referring to table 1) by following standard then:
Situation 1: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates to occur a peak at 90 ℃ ± 1 ℃, locates there is not the peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is non-deletion type (α α/α α or α α/α
Tα).
Situation 2: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates to occur a peak at 90 ℃ ± 1 ℃, locates to occur a peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is--SEA/ α α or--SEA/ α
Tα.
Situation 3: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates there is not the peak at 90 ℃ ± 1 ℃, locates to occur a peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is--SEA/--SEA.
Situation 4: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates to occur a peak at 90 ℃ ± 1 ℃, does not have the peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/ α α.
Situation 5: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production does not have the peak at 90 ℃ ± 1 ℃, locates there is not the peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/-α
3.7
Situation 6: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates to occur a peak at 90 ℃ ± 1 ℃, locates there is not the peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is-α
4.2/ α α.
Situation 7: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates there is not the peak at 90 ℃ ± 1 ℃, locates there is not the peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is-α
4.2/-α
4.2
Situation 8: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates there is not the peak at 90 ℃ ± 1 ℃, locates to occur a peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/--SEA.
Situation 9: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production locates there is not the peak at 90 ℃ ± 1 ℃, locates to occur a peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates there is not the peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is-α
4.2/--SEA.
Situation 10: if the system of employing first is carried out pcr amplification, the solubility curve of amplified production does not have the peak at 90 ℃ ± 1 ℃, locates there is not the peak at 85 ℃ ± 1 ℃; Employing system second is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 83 ℃ ± 1 ℃; Employing system third is carried out pcr amplification, and the solubility curve of amplified production locates to occur a peak at 82.5 ℃ ± 1 ℃; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/-α
4.2
Whether table 1 is the standard of missing gene type according to the specific peak interpretation sample in the solubility curve
The PCR product is carried out sex change high performance liquid chromatography (DHPLC) analysis at 50 ℃, carries out interpretation as a result and make diagnosis (referring to table 2) by following standard then:
Situation 1: if the system of employing first is carried out pcr amplification, a peak appears in amplified production at 5 ± 0.1min place, do not have the peak at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and amplified production does not have the peak at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and amplified production does not have the peak at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is non-deletion type (α α/α α or α α/α
Tα).
Situation 2: if the system of employing first is carried out pcr amplification, a peak appears in amplified production at 5 ± 0.1min place, a peak occurs at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and amplified production does not have the peak at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and amplified production does not have the peak at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is--SEA/ α α or--SEA/ α
Tα.
Situation 3: if the system of employing first is carried out pcr amplification, amplified production does not have the peak at 5 ± 0.1min place, a peak occurs at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and amplified production does not have the peak at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and amplified production does not have the peak at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is--SEA/--SEA.
Situation 4: if the system of employing first is carried out pcr amplification, a peak appears in amplified production at 5 ± 0.1min place, do not have the peak at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and a peak appears in amplified production at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and amplified production does not have the peak at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/ α α.
Situation 5: if the system of employing first is carried out pcr amplification, amplified production does not have the peak at 5 ± 0.1min place, does not have the peak at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and a peak appears in amplified production at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and amplified production does not have the peak at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/-α
3.7
Situation 6: if the system of employing first is carried out pcr amplification, a peak appears in amplified production at 5 ± 0.1min place, do not have the peak at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and amplified production does not have the peak at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and a peak appears in amplified production at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is-α
4.2/ α α.
Situation 7: if the system of employing first is carried out pcr amplification, amplified production does not have the peak at 5 ± 0.1min place, does not have the peak at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and amplified production does not have the peak at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and a peak appears in amplified production at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is-α
4.2/-α
4.2
Situation 8: if the system of employing first is carried out pcr amplification, amplified production does not have the peak at 5 ± 0.1min place, a peak occurs at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and a peak appears in amplified production at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and amplified production does not have the peak at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/--SEA.
Situation 9: if the system of employing first is carried out pcr amplification, amplified production does not have the peak at 5 ± 0.1min place, a peak occurs at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and amplified production does not have the peak at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and a peak appears in amplified production at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is-α
4.2/--SEA.
Situation 10: if the system of employing first is carried out pcr amplification, amplified production does not have the peak at 5 ± 0.1min place, does not have the peak at 1 ± 0.1min place; Employing system second is carried out pcr amplification, and a peak appears in amplified production at 2.5 ± 0.1min place; Employing system third is carried out pcr amplification, and a peak appears in amplified production at 2.3 ± 0.1min place; At the α-Zhu Danbai gene, the genotype of sample is-α
3.7/-α
4.2
Whether table 2 is the standard of missing gene type according to the specific peak interpretation sample in the DHPLC collection of illustrative plates
The PCR product of system first is carried out sex change high performance liquid chromatography (DHPLC) analysis at 63.8-64.0 ℃, carries out interpretation as a result and make diagnosis (referring to table 3) by following standard then:
Situation 1: if amplified production is unimodal one of 4.8-4.9min place appearance, do not have the peak, do not have the peak, do not have the peak at the 4.5-4.6min place at the 4.2-4.4min place at the 4.1-4.2min place; At the α-Zhu Danbai gene, the genotype of sample is not mutated type (α α/α α).
Situation 2: bimodal if amplified production is bimodal one of 4.8-4.9min place appearance one of 4.1-4.2min place appearance, there is not the peak at the 4.2-4.4min place, there is not the peak at the 4.5-4.6min place; At the α-Zhu Danbai gene, the genotype of sample is α
CSα/α α.
Situation 3: if amplified production is unimodal one of 4.8-4.9min place appearance, do not have the peak, do not have the peak at the 4.2-4.4min place at the 4.1-4.2min place, bimodal one of 4.5-4.6min place appearance; At the α-Zhu Danbai gene, the genotype of sample is α
QSα/α α.
Situation 4: if amplified production does not have the peak at the 4.8-4.9min place, do not have the peak, occur a triplet at the 4.2-4.4min place, do not have the peak at the 4.5-4.6min place at the 4.1-4.2min place; At the α-Zhu Danbai gene, the genotype of sample is α
WSα/α α.
Whether table 3 is the standard of mutator gene type according to the specific peak interpretation sample in the DHPLC collection of illustrative plates
Use test kit of the present invention, can carry out somatotype, thereby whether judgement sample is which kind of a thalassemia patient a thalassemia patient and sample belong to the entrained α-Zhu Danbai encoding gene of sample.Use test kit of the present invention and detect, have that speed is fast, automatization, high-throughput (can carry out a plurality of samples simultaneously), need not to use advantage such as contaminative medicine.The present invention has far-reaching social effect.
Description of drawings
Fig. 1 is the solubility curve of the amplified production of first group of sample and the 4th group of sample system first.
Fig. 2 is the solubility curve of the amplified production of second group of sample system first.
Fig. 3 is the solubility curve of the amplified production of the 3rd group of sample, the 6th group of sample and the 8th group of sample system first.
Fig. 4 is the solubility curve of the amplified production of first group of sample, second group of sample, the 4th group of sample and the 5th group of sample system second.
Fig. 5 is the solubility curve of the amplified production of first group of sample, second group of sample, the 6th group of sample and the 7th group of sample system third.
Fig. 6 is the DHPLC spectrum of the amplified production of second group of sample system first.
Fig. 7 is the DHPLC spectrum of the amplified production of the 6th group of sample.Among Fig. 7,7-A is the amplified production of system first, and 7-B is the amplified production of system third.
Fig. 8 is the DHPLC spectrum of the amplified production of the 4th group of sample.Among Fig. 8,8-A is the amplified production of system first, and 8-B is the amplified production of system second.
Fig. 9 is the DHPLC spectrum of the amplified production of the 11 group of sample.
Figure 10 is the DHPLC spectrum of the amplified production of the 12 group of sample.
Figure 11 is the DHPLC spectrum of the amplified production of the 13 group of sample.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Used sample is the DNA that extracts from the anticoagulation cirumferential blood white corpuscle in following examples, has 39 samples among the embodiment, is divided into 13 groups, and is specific as follows:
At the α-Zhu Danbai gene, the genotype of first group of sample is α α/α α (comprising 3 sample: 1-1,1-2 and 1-3), the genotype of second group of sample is--SEA/ α α (comprising 3 sample: 2-1,2-2 and 2-3), the genotype of the 3rd group of sample is--SEA/--SEA (comprising 3 sample: 3-1,3-2 and 3-3), the genotype of the 4th group of sample is-α
3.7/ α α (comprising 3 sample: 4-1,4-2 and 4-3), the genotype of the 5th group of sample is-α
3.7/-α
3.7(comprising 3 sample: 5-1,5-2 and 5-3), the genotype of the 6th group of sample is-α
4.2/ α α (comprising 3 sample: 6-1,6-2 and 6-3), the genotype of the 7th group of sample is-α
4.2/-α
4.2(comprising 3 sample: 7-1,7-2 and 7-3), the genotype of the 8th group of sample is-α
3.7/--SEA (comprising 3 sample: 8-1,8-2 and 8-3), the genotype of the 9th group of sample is-α
4.2/--SEA (comprising 3 sample: 9-1,9-2 and 9-3), the genotype of the tenth group of sample is-α
3.7/-α
4.2(comprising 3 sample: 10-1,10-2 and 10-3), the genotype of the 11 group of sample is α
CSα/α α (comprising 3 sample: 11-1,11-2 and 11-3), the genotype of the 12 group of sample is α
QSα/α α (comprising 3 sample: 12-1,12-2 and 12-3), the genotype of the 13 group of sample is α
WSα/α α (comprising 3 sample: 13-1,13-2 and 13-3).
The preparation of embodiment 1, globin gene disappearance and sudden change test kit
The test kit of each person-portion is composed of the following components:
The component of system first (22ul system):
Primer 1:5 '-TGC GGG CCT GGG CCG CA-3 ', concentration is 0.6ug/ul, 0.6ul;
Primer 2: 5 '-CCA TCG GGC AGG AGG AAC-3 ', concentration is 0.6ug/ul, 0.6ul;
Primer 3:5 '-TCG CGG CCT GGG GTT CAC TT-3 ', concentration is 0.6ug/ul, 0.4ul;
Primer 4:5 '-TGG ACT TAA GTG ATC CTC CTG-3 ', concentration is 0.6ug/ul, 0.6ul;
2.5ul10 * buffer (contains NH
4Cl), 2.0ul dNTP, 2.5ul MgCl
2, 5.0ul trimethyl-glycine (Betaine), 0.5ul SYBR Green1,1ul1U/ul Gold-Taq enzyme, 6.3ul dH
2O.
The component of system second (22ul system):
Primer 5:5 '-CTT TCC CTA CCC AGA GCC AGG-3 ', concentration is 0.06ug/ul, 1.0ul;
Primer 6:5 '-GCC CAA GGG GCA AGA AGC ATG-3 ', concentration is 0.10ug/ul, 0.4ul;
2.5ul 10 * buffer (contains NH
4Cl), 2.5ul 7-deaza-dGTP/dNTP (7-deaza-dGTP and dGTP equivalent), 0.02ul beta-mercaptoethanol, 1.5ul DMSO, 2.5ul trimethyl-glycine (Betaine), 0.5ulSYBR Greenl, 1ul2U/ul Gold-Taq enzyme, 10.08ul dH
2O.
The component of system third (22ul system):
Primer 7:5 '-TTT GAA TGA AGT CCG AGT AGG CAG-3 ', concentration is 0.10ug/ul, 1.0ul;
Primer 8:5 '-CCC TGG GTG TCC AGG AGC AAG CC-3 ', concentration is 0.10ug/ul, 0.3ul;
2.5ul 10 * buffer (contains NH
4Cl), 2.5ul 7-deaza-dGTP/dNTP (7-deaza-dGTP and dGTP equivalent), 0.02ul beta-mercaptoethanol, 1.0ul DMSO, 2.5ul trimethyl-glycine (Betaine), 0.5ulSYBR Greenl, 1ul2U/ul Plus-Taq enzyme, 10.68ul dH
2O.
The application of embodiment 2, globin gene disappearance and sudden change test kit
The test kit of Application Example 1 preparation detects 13 groups of (39 parts) samples.The detection step of each sample is as follows:
One, pcr amplification
Extracting the genomic dna of sample, is template with the 3ul genomic dna, carries out the Realtime-PCR reaction (ABI-5700 PCR in real time amplification instrument) of following three individual system respectively:
System first: carry out the PCR reaction with the system first.
System second: carry out the PCR reaction with system second.
System third: carry out the PCR reaction with system third.
The PCR reaction conditions of system first: 94 ℃ of 10min → 94 ℃ 20s, 64 ℃ of 30s, 72 ℃ of 40s → 94 ℃ 20s, 63 ℃ of 30s, 72 ℃ of 40s → 94 ℃ 20s, 62 ℃ of 30s, 72 ℃ of 40s → 94 ℃ 20s, 61 ℃ of 30s, 72 ℃ of 40s; → 72 ℃ of 6min of 34 circulations.
The PCR reaction conditions of system second and system third: 94 ℃ of 10min → 94 ℃ 20s, 65 ℃ of 1min, 72 ℃ of 2min → 94 ℃ 20s, 64 ℃ of 1min, 72 ℃ of 2min → 94 ℃ 20s, 63 ℃ of 1min, 72 ℃ of 2min → 94 ℃ 20s, 62 ℃ of 1min, 72 ℃ of 2min → 94 ℃ 20s, 61 ℃ of 1min, 72 ℃ of 2min; → 72 ℃ of 6min of 34 circulations.
Two, solubility curve analysis and dhplc analysis
(1) solubility curve analysis
To directly carry out 60 ℃ to 95 ℃ solubility curve analysis with first group to the tenth group the sample that the amplification of ABI-5700 PCR in real time detector obtains respectively.
3 samples in every group all show identical or akin solubility curve.The results are shown in Table 4.
Table 4 is by the genotype result of solubility curve analyzing and testing sample
First group of sample: the solubility curve of the amplified production of system first is seen the curve 1 of Fig. 1, and the solubility curve of the amplified production of system second is seen the curve 3 of Fig. 4, and the solubility curve of the amplified production of system third is seen the curve 3 of Fig. 5.
Second group of sample: the solubility curve of the amplified production of system first sees that (curve 1 is sample 2-1 for the curve 1 of Fig. 2 and curve 2, curve 2 is sample 2-2), the solubility curve of the amplified production of system second is seen the curve 4 of Fig. 4, and the solubility curve of the amplified production of system third is seen the curve 4 of Fig. 5.
The 3rd group of sample: the solubility curve of the amplified production of system first is seen the curve 1 of Fig. 3.
The 4th group of sample: the solubility curve of the amplified production of system first is seen the curve 2 of Fig. 1, and the solubility curve of the amplified production of system second is seen the curve 1 of Fig. 4.
The 5th group of sample: the solubility curve of the amplified production of system second is seen the curve 2 of Fig. 4.
The 6th group of sample: the solubility curve of the amplified production of system first is seen the curve 3 of Fig. 3, and the solubility curve of the amplified production of system third is seen the curve 1 of Fig. 5.
The 7th group of sample: the solubility curve of the amplified production of system third is seen the curve 2 of Fig. 5.
The 8th group of sample: the solubility curve of the amplified production of system first is seen the curve 2 of Fig. 3.
(2) DHPLC analyzes
1, sex change high performance liquid chromatography (50 ℃) is analyzed
PCR product with three individual system of first group to the tenth group sample carries out sex change high performance liquid chromatography (DHPLC) analysis at 50 ℃ respectively.
3 samples in every group all show identical or akin curve.The results are shown in Table 5.
Table 5 is by genotype result's (50 ℃) of DHPLC analyzing and testing sample
The DHPLC spectrum of the amplified production of second group of sample system first is seen Fig. 6.
The DHPLC spectrum of the amplified production of the 6th group of sample is seen Fig. 7.Among Fig. 7,7-A is the amplified production of system first, and 7-B is the amplified production of system third.
The DHPLC spectrum of the amplified production of the 4th group of sample is seen Fig. 8.Among Fig. 8,8-A is the amplified production of system first, and 8-B is the amplified production of system second.
2, sex change high performance liquid chromatography (63.8 ℃) is analyzed
PCR product with the system first of first group, the 11 group, the 12 group, the 13 group sample carries out sex change high performance liquid chromatography (DHPLC) analysis at 63.8-64.0 ℃ respectively.
3 samples in every group all show identical or akin curve.The results are shown in Table 6.
Table 6 is by genotype result's (63.8 ℃) of DHPLC analyzing and testing sample
The DHPLC spectrum of the amplified production of the 11 group of sample is seen Fig. 9.The DHPLC spectrum of the amplified production of the 12 group of sample is seen Figure 10.The DHPLC spectrum of the amplified production of the 13 group of sample is seen curve 1 and the curve 2 of Figure 11.
Sequence table
＜110〉Beijing Chaoyang Hospital Attached to Capital Medical Univ.
＜120〉a kind of test kit that detects α-Zhu Danbai genetically deficient and sudden change
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Claims (10)
1, a kind of test kit by solubility curve analysis and/or sex change high performance liquid chromatography detection α-Zhu Danbai genetically deficient and sudden change comprises as next group primer:
Primer 1 is incorporated into second intron of α-Zhu Danbai gene cluster α 2 genes and compares the sequence place that lacks 7 nucleotide pairs near the 3rd exon with α 1; Primer 2 is incorporated into α-Zhu Danbai gene cluster α 2 gene terminator codons 3 ' catchment;
Primer 3 and primer 4 are incorporated into the genomic dna of the upstream and downstream in disappearance zone, α-Zhu Danbai gene cluster South East Asia respectively.
2, test kit as claimed in claim 1 is characterized in that: described test kit also comprises following two groups of primers:
Second group of primer is made up of primer 5 and primer 6, and primer 5 and primer 6 are incorporated into α-Zhu Danbai gene cluster-α respectively
3.7The genomic dna of the upstream and downstream in disappearance zone;
The 3rd group of primer is made up of primer 7 and primer 8, and primer 7 and primer 8 are incorporated into α-Zhu Danbai gene cluster-α respectively
4.2The genomic dna of the upstream and downstream in disappearance zone.
3, test kit as claimed in claim 2, it is characterized in that: the sequence of described primer 1 is shown in the sequence 1 of sequence table, the sequence of described primer 2 is shown in the sequence 2 of sequence table, the sequence of described primer 3 is shown in the sequence 3 of sequence table, the sequence of described primer 4 is shown in the sequence 4 of sequence table, the sequence of described primer 5 is shown in the sequence 5 of sequence table, the sequence of described primer 6 is shown in the sequence 6 of sequence table, the sequence of described primer 7 is shown in the sequence 7 of sequence table, and the sequence of described primer 8 is shown in the sequence 8 of sequence table.
4, test kit as claimed in claim 3 is characterized in that: described test kit also comprises nonspecific embedding fluorescence dye.
5, test kit as claimed in claim 4 is characterized in that: described fluorescence dye is SYBR Green I.
6, test kit as claimed in claim 5 is characterized in that: described test kit also comprises the conventional reagent of real-time fluorescence quantitative PCR instrument or sex change high performance liquid chromatograph and Realtime-PCR.
7, test kit as claimed in claim 6 is characterized in that: described test kit is applied to crowd's examination, gene diagnosis and the prenatal gene diagnosis of α-Di Zhonghaipinxue.
8, comprise the application of primer sets in preparing the test kit that detects α-Zhu Danbai genetically deficient and sudden change by solubility curve analysis and/or sex change high performance liquid chromatography of primer 1, primer 2, primer 3 and primer 4;
Described primer 1 is incorporated into second intron of α-Zhu Danbai gene cluster α 2 genes and compares the sequence place that lacks 7 nucleotide pairs near the 3rd exon with α 1; Described primer 2 is incorporated into α-Zhu Danbai gene cluster α 2 gene terminator codons 3 ' catchment;
Described primer 3 and primer 4 are incorporated into the genomic dna of the upstream and downstream in disappearance zone, α-Zhu Danbai gene cluster South East Asia respectively.
9, application as claimed in claim 8 is characterized in that: described primer sets also comprises primer 5, primer 6, primer 7 and primer 8;
Described primer 5 and primer 6 are incorporated into α-Zhu Danbai gene cluster-α respectively
3.7The genomic dna of the upstream and downstream in disappearance zone;
Described primer 7 and primer 8 are incorporated into α-Zhu Danbai gene cluster-α respectively
4.2The genomic dna of the upstream and downstream in disappearance zone.
10, application as claimed in claim 9, it is characterized in that: the sequence of described primer 1 is shown in the sequence 1 of sequence table, the sequence of described primer 2 is shown in the sequence 2 of sequence table, the sequence of described primer 3 is shown in the sequence 3 of sequence table, the sequence of described primer 4 is shown in the sequence 4 of sequence table, the sequence of described primer 5 is shown in the sequence 5 of sequence table, the sequence of described primer 6 is shown in the sequence 6 of sequence table, the sequence of described primer 7 is shown in the sequence 7 of sequence table, and the sequence of described primer 8 is shown in the sequence 8 of sequence table.
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| CN2008102278919A CN101413032B (en) | 2008-12-02 | 2008-12-02 | Reagent kit for detecting deletion and mutation of alpha-globin gene |
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| CN2008102278919A CN101413032B (en) | 2008-12-02 | 2008-12-02 | Reagent kit for detecting deletion and mutation of alpha-globin gene |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344925A (en) * | 2011-10-20 | 2012-02-08 | 昆明金域医学检验所有限公司 | Depletion alpha thalassemia-2 gene and assay kit and detection method thereof |
| CN102936631A (en) * | 2012-12-04 | 2013-02-20 | 亚能生物技术（深圳）有限公司 | Gene detection kit for Hong Kong alpha-thalassemia |
| CN104830964A (en) * | 2015-02-03 | 2015-08-12 | 赣南医学院第一附属医院 | Detection method of Southeast Asian thalassemia |
| CN105154579A (en) * | 2015-10-26 | 2015-12-16 | 钦州市妇幼保健院 | Reagent kit for rapidly detecting -alpha21.9 deletion type thalassemia alleles |
| CN111909990A (en) * | 2020-08-28 | 2020-11-10 | 亚能生物技术（深圳）有限公司 | Fluorescent PCR detection method for simultaneously detecting deletion mutation and point mutation of gene by single tube |
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2008
- 2008-12-02 CN CN2008102278919A patent/CN101413032B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344925A (en) * | 2011-10-20 | 2012-02-08 | 昆明金域医学检验所有限公司 | Depletion alpha thalassemia-2 gene and assay kit and detection method thereof |
| CN102344925B (en) * | 2011-10-20 | 2014-04-30 | 昆明金域医学检验所有限公司 | Depletion alpha thalassemia-2 gene and assay kit and PCR sequencing method thereof |
| CN102936631A (en) * | 2012-12-04 | 2013-02-20 | 亚能生物技术（深圳）有限公司 | Gene detection kit for Hong Kong alpha-thalassemia |
| CN102936631B (en) * | 2012-12-04 | 2014-03-19 | 亚能生物技术（深圳）有限公司 | Gene detection kit for Hong Kong alpha-thalassemia |
| CN104830964A (en) * | 2015-02-03 | 2015-08-12 | 赣南医学院第一附属医院 | Detection method of Southeast Asian thalassemia |
| CN105154579A (en) * | 2015-10-26 | 2015-12-16 | 钦州市妇幼保健院 | Reagent kit for rapidly detecting -alpha21.9 deletion type thalassemia alleles |
| CN111909990A (en) * | 2020-08-28 | 2020-11-10 | 亚能生物技术（深圳）有限公司 | Fluorescent PCR detection method for simultaneously detecting deletion mutation and point mutation of gene by single tube |
| CN111909990B (en) * | 2020-08-28 | 2023-11-28 | 亚能生物技术（深圳）有限公司 | Fluorescent PCR detection method for simultaneously detecting deletion mutation and point mutation of gene by single tube |
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| CN101413032B (en) | 2012-08-22 |
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