CN105176995A - Hydrolysis probe method based kit capable of simultaneously detecting deletion -alpha<21.9> and alpha2 variation of thalassemia - Google Patents

Abstract

The invention discloses a hydrolysis probe method based kit capable of simultaneously detecting deletion -alpha<21.9> and an alpha2 variant of thalassemia. The alpha2 variant is a variant genotype of a gene alpha2 in an alpha-globin gene cluster (NG_000006.1). The genotype is described as HBA2:c.301-24delinsCTCGGCCC. The kit comprises a pair of primers capable of amplifying the characteristic sequences of the allele -alpha<21.9>, a pair of primers capable of amplifying the characteristic sequence of the variant allele alpha2, a pair of primers capable of amplifying reference gene GAPDH fragments, a probe for specifically detecting the -alpha<21.9> amplification primers, a probe for specifically detecting the variant allele alpha2 amplification primers and a probe for specifically detecting the GAPDH fragment amplification primers. The kit can directly complete detection of the alpha2 variant and the -alpha<21.9> genotype through single-pipe single reaction.

Description

Thalassemia-α can be detected based on hydrolysis probes method simultaneously 21.9the test kit that absence type and α 2 make a variation

Technical field

The present invention relates to technical field of medical detection, be specifically related to a kind ofly can detect thalassemia-α based on hydrolysis probes method simultaneously ^21.9the test kit that absence type and α 2 make a variation.

Background technology

Thalassemia also claims globin dyssynthesis anaemia, is called for short poor.Thalassemia is one of modal monogenic inheritance disease in Guangxi province, and the poor kind in common ground has α-ground poor poor with β-ground.In Chinese population, what the poor genotype in α-ground was common is absence type-- ^sEA,-α ^3.7with-α ^4.2.2013, healthcare hospital for women & children of Qinzhou City detected the rarely poor genotype of a kind of called after ' the poor absence type in Qiezhou type α ground ', be the disappearance of the DNA sequence dna of one section of 21.9kb in alpha globin gene bunch (NG_000006.1) due to this genotypic the Molecular Biology Mechanism, be therefore abbreviated as-α ^21.9(LongJ, YanS, LaoK, PangW, YeX, SunL.Thediagnosisandmolecularanalysisofanovel21.9kbdelet ion (Qinzhoutypedeletion) causing α+thalassemia.BloodCellsMolDis.2014; 52 (4): 225-9.).In addition, in the research process of applicant, also find the mutant gene type of α 2 gene in a kind of novel α-globin gene cluster (NG_000006.1), this genotype is described to HBA2:c.301-24delinsCTCGGCCC.Applicant also finds that this genotype has certain distribution probability in Guangxi, and for detecting these two kinds of genotype, needing to set up one can simultaneously and rapid detection HBA2:c.301-24delinsCTCGGCCC allelotrope-α ^21.9allelic test kit.

Summary of the invention

The technical problem to be solved in the present invention is to provide and a kind ofly can detects thalassemia-α based on hydrolysis probes method simultaneously ^21.9the test kit that absence type and α 2 make a variation.This test kit can realize the reaction of single tube single and directly complete HBA2:c.301-24delinsCTCGGCCC and-α ^21.9allelic detection, and there is susceptibility highly, stability and accuracy, and higher specificity.

Of the present inventionly can detect thalassemia-α based on hydrolysis probes method simultaneously ^21.9the test kit that absence type and α 2 make a variation, comprises amplimer and fluorescent probe, wherein:

Described α 2 makes a variation as the mutant gene type of α 2 gene in α-globin gene cluster (NG_000006.1), and this genotype is described to HBA2:c.301-24delinsCTCGGCCC, and sequence is specially:

gggttgcgggaggtgtagcgcaggcggcggctgcgggcctgggccCTCGGCCCcactgaccctcttctctgcacagctcctaagccactgcctgctggtgaccctggccgcccacctccccgccgagttcacccctgcggtgcacgcctccctggacaagttcctggcttctgtgagcaccgtgctgacctccaaataccgttaagctggagcctcggtagccgttcctcctgcccgctgggcctcccaacgggccctcctcccctccttgcaccggcccttcctggtctttgaataaagtctgagtgggcagcagcctgtgtgtgcctgggttctctctatcccggaatgtgccaacaatggaggtgtttacctgtctcagaccaagga；

Described amplimer is :-α in the α-globin gene cluster that can increase for a pair ^21.9primer 2 1.9-F and 21.9-R of allelic characteristic sequence, primer A2V-F and A2V-R of the allelic characteristic sequence of HBA2:c.301-24delinsCTCGGCCC in the α-globin gene cluster that can increase for a pair, and primer GAPDH-F and GAPDH-R of the reference gene GAPDH fragment that can increase for a pair;

Described fluorescent probe is: the fluorescent probe 21.9-Prob of a specific detection 21.9-F and 21.9-R amplified production,, a fluorescent probe A2V-Prob for specific detection A2V-F and A2V-R amplified production, the fluorescent probe GAPDH-Prob of a specific detection GAPDH-F and GAPDH-R amplified production;

Wherein:

-α in the described α-globin gene cluster that can increase ^21.9in the primer pair of allelic characteristic sequence,

21.9-F：5’-GGAGCTTTTCCTTCCCTGGAACG-3’；

21.9-R：5’-TGTGGTTGGAGAATGGAGGTGG-3’；

In the described α-globin gene cluster that can increase the allelic characteristic sequence of HBA2:c.301-24delinsCTCGGCCC primer pair in,

A2V-F：5’-GGGTTGCGGGAGGTGTAG-3’；

A2V-R：5’-TCCTTGGTCTGAGACAGGTA-3’；

Describedly can increase in the primer pair of reference gene GAPDH fragment,

GAPDH-F：5’-CCCCACACACATGCACTTACC-3’；

GAPDH-R：5’-CCTAGTCCCAGGGCTTTGATT-3’；

The fluorescent probe 21.9-Prob of described specific detection 21.9-F and 21.9-R amplified production is:

21.9-Prob：5’-6-FAM-CTGGGGAAGGGTGGGTGGGAATAACAGC-BHQ-1-3’；

The fluorescent probe A2V-Prob of described specific detection A2V-F and A2V-R amplified production is:

A2V-Prob：5’-6-ROX-CCTCGGCCCCACTGACCCTC-BHQ-1-3’；

The fluorescent probe GAPDH-Prob of described specific detection GAPDH-F and GAPDH-R amplified production is:

GAPDH-Prob：5’-CY5-AAAGAGCTAGGAAGGACAGGCAACTTGGC-BHQ-2-3’。

In fluorescent probe of the present invention, FAM refers to Fluoresceincarboxylic acid, and ROX refers to carboxy-X-rhodamine, and CY5 refers to that cyanine dyes molecule 5, BHQ-1 and BHQ-2 refer to fluorescent quenching group.

Test kit of the present invention, also comprises component conventional and necessary in some available reagent boxes, as damping fluid, enzyme liquid, MgCl ₂and dNTP.Particularly, enzyme liquid is Taq polymerase systems, comprises the warm start enzyme system etc. that can be used for hydrolysis probes method; Described damping fluid is conventional PCR damping fluid.When enzyme liquid select to adopt health for produce in century GoldStarTaqDNAPolymerase time, damping fluid is then preferably the damping fluid supporting with GoldStarTaqDNAPolymerase.

Adopt mentioned reagent box rapid detection HBA2:c.301-24delinsCTCGGCCC and-α ^21.9allelic method, comprises the following steps:

1) extracting sample genomic dna, prepares DNA profiling;

2) prepare reaction system, be specially:

By primer 2 1.9-F, 21.9-R, A2V-F, A2V-R, GAPDH-F and GAPDH-R, fluorescent probe 21.9-Prob, A2V-Prob and GAPDH-Prob; And PCR damping fluid, enzyme liquid, MgCl2, dNTP, water and DNA profiling are mixed with reaction system;

3) pattern detection: respectively the reaction system of preparation is carried out pcr amplification, records the cycle index Cq (size of Cq value can reflect institute's detection template number number) experienced when fluorescent signal in each PCR reaction tubes arrives the thresholding of setting;

4) data analysis and result judge: the analysis software sentence read result carried according to real-time fluorescence quantitative PCR instrument, and when CY5 passage has Cq value reading, then this sample is normally increased;

Have the prerequisite of reading at CY5 passage under, if ROX passage has reading, then represent that this sample carries HBA2:c.301-24delinsCTCGGCCC allelotrope;

Have the prerequisite of reading at CY5 passage under, if ROX passage does not have Cq value reading, then represent that this sample does not carry HBA2:c.301-24delinsCTCGGCCC allelotrope;

Have the prerequisite of reading at CY5 passage under, if FAM passage has Cq value reading, then represent that this sample carries-α ^21.9allelotrope;

Have the prerequisite of reading at CY5 passage under, if FAM passage does not have Cq value reading, then represent that this sample does not carry-α ^21.9allelotrope.

The step 1 of aforesaid method) in, adopt existing ordinary method to prepare DNA profiling.

The step 2 of aforesaid method) in, in reaction system, the concentration of each component is preferably: DNA:1 ~ 3ng/ μ L to be detected; Each primer: 0.3 ~ 0.5 μm of ol/L; Each fluorescent probe concentration is 0.3 ~ 0.5 μm of ol/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 ~ 12 minutes, then 95 DEG C 15 ~ 30 seconds, 60 DEG C of annealing 50 ~ 70 seconds, 39 ~ 49 circulations, in 60 DEG C of annealing steps end collection fluorescent signals.

Compared with prior art, feature of the present invention is:

1, test kit of the present invention can realize the reaction of single tube single and directly complete HBA2:c.301-24delinsCTCGGCCC and-α ^21.9allelic detection, and there is susceptibility highly, stability and accuracy, and higher specificity.

2, without the need to open pipe operation, the possibility that laboratory PCR primer is polluted is reduced to the utmost;

3, adopt the experimental program of hydrolysis probes fewer than existing additive method required time, not high to equipment requirements yet.

Accompanying drawing explanation

Fig. 1 is the amplification curve of 1# sample in the embodiment of the present invention 1;

Fig. 2 is the amplification curve of 2# sample in the embodiment of the present invention 1;

Fig. 3 is the amplification curve of 3# sample in the embodiment of the present invention 1.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail, and to understand content of the present invention better, but the present invention is not limited to following examples.

Embodiment 1: adopt the detected result of test kit of the present invention in known type sample

1, the composition of test kit:

(1)-α in the α-globin gene cluster that can increase ^21.9primer pair 21.9-F and 21.9-R of allelic characteristic sequence:

21.9-F：5’-GGAGCTTTTCCTTCCCTGGAACG-3’(SEQIDNO：1)；

21.9-R：5’-TGTGGTTGGAGAATGGAGGTGG-3’(SEQIDNO：2)；

(2) can amplify alpha globin gene cluster of HBA2: c. 301-24 delinsctcggccc allele (described HBA2: c. 301-24 delinsctcggccc gene sequence specific as follows:gggttgcgggaggtgtagcgcaggcggcggctgcgggcctgggccCTCGGCCCcactgaccctcttctctgcacagctcctaagccactgcctgctggtgaccctggccgcccacctccccgccgagttcacccctgcggtgcacgcctccctggacaagttcctggcttctgtgagcaccgtgctgacctccaaataccgttaagctggagcctcggtagccgttcctcctgcccgctgggcctcccaacgggccctcctcccctccttgcaccggcccttcctggtctttgaataaagtctgagtgggcagcagcctgtgtgtgcctgggttctctctatcccggaatgtgccaacaatggaggtgtttacctgtctcagaccaagga(SEQIDNO：3) the characteristics of sequence primers for A2V -f and A2V - R:

A2V-F:5’-GGGTTGCGGGAGGTGTAG-3’(SEQIDNO：4)；

A2V-R:5’-TCCTTGGTCTGAGACAGGTA-3’(SEQIDNO：5)；

(3) GAPDH-F and GAPDH-R in the primer pair of the reference gene GAPDH fragment that can increase:

GAPDH-F:5’-CCCCACACACATGCACTTACC-3’(SEQIDNO：6)；

GAPDH-R:5’-CCTAGTCCCAGGGCTTTGATT-3’(SEQIDNO：7)；

(4) the fluorescent probe 21.9-Prob of specific detection 21.9-F and 21.9-R amplified production is:

21.9-Prob:5’-6-FAM-CTGGGGAAGGGTGGGTGGGAATAACAGC-BHQ-1-3’(SEQIDNO：8)；

(5) the fluorescent probe A2V-Prob of specific detection A2V-F and A2V-R amplified production is:

A2V-Prob:5’6-ROX-CCTCGGCCCCACTGACCCTC-3’BHQ-1(SEQIDNO：9)。

(6) the fluorescent probe GAPDH-Prob of specific detection GAPDH-F and GAPDH-R amplified production is:

GAPDH-Prob:5’-CY5-AAAGAGCTAGGAAGGACAGGCAACTTGGC-BHQ2-3’(SEQIDNO：10)。

The sequence of above-mentioned reference gene GAPDH fragment is see (ZimmermannB, HolzgreveW, WenzelF, HahnS.Novelreal-timequantitativePCRtestfortrisomy21.Clin Chem.2002Feb; 48 (2): 362-3.), specific as follows:

CCCCACACACATGCACTTACCTGTGCTCCCACTCCTGATTTCTGGAAAAGAGCTAGGAAGGACAGGCAACTTGGCAA(SEQIDNO：11)。

(7) other moiety:

GoldStarTaqDNAPolymerase, be all century purchased from health with the supporting damping fluid of GoldStarTaqDNAPolymerase and dNTPs, MgCl ₂purchased from LifeTechnology.

PCR reaction system is prepared by following table 1:

Table 1:(mM represents mmol/L, μM expression μm ol/L)

2, implementation method:

It is Bio-Rad real time thermocycler CFX96 that PCR reacts instrument.PCR response procedures is: 95 DEG C of denaturations 10 minutes, then 95 DEG C 15 seconds, 60 DEG C of annealing 60 seconds, 40 circulations, in 60 DEG C of annealing steps end collection fluorescent signals.

Sample process: adopt Lab-AidDNAminiextractionkid (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 method to extract.

Pattern detection: (wherein 1 part is known to HBA2:c.301-24delinsCTCGGCCC genotype sample to be checked (being called for short 1# sample) by 2 parts, sample to be checked for known type; Another part is known to-α ^21.9genotype sample to be checked (being called for short 2# sample)); and 1 part of normal genotype (α α/α α, N/N) sample (being called for short 3# sample), according to above-mentioned reaction system and response procedures; augmentation detection is carried out, record Cq value on quantitative real time PCR Instrument.

3, samples sources: all sample standard deviations source conventional Gap-PCR technology of hanging oneself determines genotypic DNA sample.

4, data analysis and result judge: the analysis software sentence read result carried according to real-time fluorescence PCR instrument.If CY5 passage has Cq value reading, then represent that this sample is normally increased;

Have the prerequisite of reading at CY5 passage under, if ROX passage has reading, then represent that this sample carries HBA2:c.301-24delinsCTCGGCCC allelotrope;

Have the prerequisite of reading at CY5 passage under, if ROX passage does not have Cq value reading, then represent that this sample does not carry HBA2:c.301-24delinsCTCGGCCC allelotrope;

Have the prerequisite of reading at CY5 passage under, if FAM passage has Cq value reading, then represent that this sample carries-α ^21.9allelotrope;

Have the prerequisite of reading at CY5 passage under, if FAM passage does not have Cq value reading, then represent that this sample does not carry-α ^21.9allelotrope.

Analyze after testing, the CY5 passage of 1# sample has Cq value and ROX passage has Cq value (as shown in Figure 1, Cq value reading as shown in Table 2 for amplification curve), represents that 1# sample carries HBA2:c.301-24delinsCTCGGCCC allelotrope; The CY5 passage of 2# sample has Cq value and FAM passage has Cq value (as shown in Figure 2, Cq value reading as shown in Table 3 for amplification curve), represents that 2# sample carries-α ^21.9allelotrope; The CY5 passage of 3# sample has Cq value and ROX passage and FAM passage do not have Cq value (as shown in Figure 3, Cq value reading as shown in Table 4 for amplification curve), represents that 3# sample does not carry HBA2:c.301-24delinsCTCGGCCC allelotrope and do not carry-α yet ^21.9allelotrope.

Table 2:

Table 3:

Table 4:

Visible, adopt test kit of the present invention carry out-α ^21.9absence type and α 2 make a variation allelotrope detect time, result is accurate; And wild-type sample specific C q value nothing but, there is higher specificity.

Embodiment 2: the Detection results of test kit 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, samples sources:

Sample is 5 routine random samples (being provided by healthcare hospital for women & children of Qinzhou City), 20 ~ 200ng is diluted to distilled water, as sample to be checked), and through 1 routine wild-type sample of Gap-PCR and sequence verification, 1 example carries HBA2:c.301-24delinsCTCGGCCC allelotrope sample, and 1 example carries-α ^21.9allelotrope sample.

4, data analysis and result judge:

According to the analysis software sentence read result that real-time fluorescence quantitative PCR instrument carries; When CY5 passage has Cq value reading, then this sample is normally increased;

Have the prerequisite of reading at CY5 passage under, if ROX passage has reading, then represent that this sample carries HBA2:c.301-24delinsCTCGGCCC allelotrope;

Have the prerequisite of reading at CY5 passage under, if ROX passage does not have Cq value reading, then represent that this sample does not carry HBA2:c.301-24delinsCTCGGCCC allelotrope;

Have the prerequisite of reading at CY5 passage under, if FAM passage has Cq value reading, then represent that this sample carries-α ^21.9allelotrope;

Have the prerequisite of reading at CY5 passage under, if FAM passage does not have Cq value reading, then represent that this sample does not carry-α ^21.9allelotrope.

Table 5:

Note: NegCtrl=negative control in sample type, PosCtrl=positive control, Unkn=unknown sample type, in detected result, N/A=is without Cq value.

Result show, wild-type sample and positive sample detect Cq value and theory consistent, detect 1 example in random sample and carry HBA2:c.301-24delinsCTCGGCCC allelotrope.

Claims (2)

1. can detect thalassemia-α based on hydrolysis probes method simultaneously ^21.9the test kit that absence type and α 2 make a variation, comprises amplimer and fluorescent probe, it is characterized in that:

Described α 2 makes a variation as the mutant gene type of α 2 gene in α-globin gene cluster (NG_000006.1), and this genotype is described to HBA2:c.301-24delinsCTCGGCCC;

Described amplimer is :-α in the α-globin gene cluster that can increase for a pair ^21.9primer 2 1.9-F and 21.9-R of allelic characteristic sequence, primer A2V-F and A2V-R of the allelic characteristic sequence of HBA2:c.301-24delinsCTCGGCCC in the α-globin gene cluster that can increase for a pair, and primer GAPDH-F and GAPDH-R of the reference gene GAPDH fragment that can increase for a pair;

Described fluorescent probe is: the fluorescent probe 21.9-Prob of a specific detection 21.9-F and 21.9-R amplified production,, a fluorescent probe A2V-Prob for specific detection A2V-F and A2V-R amplified production, the fluorescent probe GAPDH-Prob of a specific detection GAPDH-F and GAPDH-R amplified production;

Wherein:

-α in the described α-globin gene cluster that can increase ^21.9in the primer pair of allelic characteristic sequence,

21.9-F：5’-GGAGCTTTTCCTTCCCTGGAACG-3’；

21.9-R：5’-TGTGGTTGGAGAATGGAGGTGG-3’；

In the described α-globin gene cluster that can increase the allelic characteristic sequence of HBA2:c.301-24delinsCTCGGCCC primer pair in,

A2V-F：5’-GGGTTGCGGGAGGTGTAG-3’；

A2V-R：5’-TCCTTGGTCTGAGACAGGTA-3’；

Describedly can increase in the primer pair of reference gene GAPDH fragment,

GAPDH-F：5’-CCCCACACACATGCACTTACC-3’；

GAPDH-R：5’-CCTAGTCCCAGGGCTTTGATT-3’；

The fluorescent probe 21.9-Prob of described specific detection 21.9-F and 21.9-R amplified production is:

21.9-Prob：5’-6-FAM-CTGGGGAAGGGTGGGTGGGAATAACAGC-BHQ-1-3’；

The fluorescent probe A2V-Prob of described specific detection A2V-F and A2V-R amplified production is:

A2V-Prob：5’-6-ROX-CCTCGGCCCCACTGACCCTC-BHQ-1-3’；

The fluorescent probe GAPDH-Prob of described specific detection GAPDH-F and GAPDH-R amplified production is:

GAPDH-Prob：5’-CY5-AAAGAGCTAGGAAGGACAGGCAACTTGGC-BHQ-2-3’。

2. test kit according to claim 1, is characterized in that: described test kit also comprises damping fluid, enzyme liquid, MgCl ₂and dNTP.