CN105154578A - Kit for quickly detecting -alpha 21.9 deletional thalassemia alleles based on fluorescent hydrolysis probe method - Google Patents

Abstract

The invention discloses a kit for quickly detecting -alpha 21.9 deletional thalassemia alleles based on a fluorescent hydrolysis probe method. The kit comprises amplification primers and a fluorescent probe, wherein the amplification primers are a pair of primers 21.9-F and 21.9-R capable of amplifying characteristics sequences of -alpha 21.9 alleles in alpha-globin gene clusters, the 21.9-F is 5'-GGAGCTTTTCCTTCCCTGGAACG-3', the 21.9-R is 5'-TGTGGTTGGAGAATGGAGGTGG-3', the fluorescent probe is a fluorescent probe 21.9-Prob for specific detection of 21.9-F and 21.9-R amplification products, and the specific 21.9-Prob is 5'-6-FAM-CTGGGGAAGGGTGGGTGGGAATAACAGC BHQ-1-3'.

Description

Based on the rapid detection-α of fluorescent hydrolysis probe method 21.9the allelic test kit of absence type thalassemia

Technical field

The present invention relates to technical field of medical detection, be specifically related to a kind of rapid detection-α based on fluorescent hydrolysis probe method ^21.9the allelic test kit of absence type thalassemia.

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 routine duties, applicant finds that this genotype has certain distribution probability in Guangxi, and genotypicly to fail to pinpoint a disease in diagnosis for reducing this, need set up one can quick-α ^21.9allelic detection kit.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of rapid detection-α based on fluorescent hydrolysis probe method ^21.9the allelic test kit of absence type thalassemia.Adopt this test kit can realize single tube single and react-α ^21.9the allelic detection of absence type thalassemia, and there is susceptibility highly, stability and accuracy, and higher specificity.

Rapid detection-α based on fluorescent hydrolysis probe method of the present invention ^21.9the allelic test kit of absence type thalassemia, comprises amplimer and fluorescent probe, it is characterized in that:

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, wherein:

21.9-F：5’-GGAGCTTTTCCTTCCCTGGAACG-3’；

21.9-R：5’-TGTGGTTGGAGAATGGAGGTGG-3’；

Described fluorescent probe is the fluorescent probe 21.9-Prob of specific detection 21.9-F and 21.9-R amplified production, is specially:

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

In fluorescent probe in mentioned reagent box, FAM refers to Fluoresceincarboxylic acid, and BHQ-1 refers to fluorescent quenching group.

Rapid detection-α based on fluorescent hydrolysis probe method of the present invention ^21.9the allelic test kit of absence type thalassemia, 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-α ^21.9the allelic method of absence type thalassemia comprises the following steps:

1) extracting sample genomic dna, prepares DNA profiling;

2) prepare reaction system, be specially:

By primer 2 1.9-F and 21.9-R, probe 21.9-Prob, 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 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 result of determination carried according to real-time fluorescence quantitative PCR instrument, when FAM passage has Cq value reading, then represents that this sample carries-α ^21.9allelotrope; When 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; Concentration and 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-α ^21.9allelic detection; P-α ^21.9allelotrope detects susceptibility, stability and the accuracy with height, and higher specificity.

2, test kit of the present invention is applied to fluorescent hydrolysis probe method detection-α ^21.9during allelotrope, without the need to open pipe operation, reduce the possibility that laboratory PCR primer is polluted to the utmost.

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

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.

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:

-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)；

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：3)。

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: detect the known type sample to be checked (being called for short 1# sample) determined by ordinary method by 1 part; and 1 part of normal genotype (α α/α α; N/N) sample (being called for short 2# 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 quantitative PCR instrument.If FAM passage has Cq value reading, then represent that this sample carries-α ^21.9allelotrope; If FAM passage does not have Cq value reading, then represent that this sample does not carry-α ^21.9allelotrope.Analyze after testing, the FAM passage of 1# sample has Cq value reading (as shown in Figure 1, Cq value reading as shown in Table 2 for amplification curve), represents that 1# sample carries-α ^21.9allelotrope; The FAM passage of 2# sample does not have Cq value (as shown in Figure 2, Cq value reading as shown in Table 3 for spectrogram), represents that 2# sample does not carry-α ^21.9allelotrope.

Table 2:

Table 3:

Visible, adopt test kit of the present invention carry out-α ^21.9when absence type thalassemia allelotrope detects, result is accurate; And wild-type sample is without non-specific amplification, 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 12 routine random samples (being provided by healthcare hospital for women & children of Qinzhou City), is diluted to 20 ~ 200ng, as sample to be checked with distilled water), and through the 2 routine wild-type samples that Gap-PCR verifies, 2 routine positive sample.

4, data analysis and result judge:

According to the analysis software sentence read result that real-time fluorescence quantitative PCR instrument carries.If FAM passage has Cq value reading, then represent that this sample carries-α ^21.9allelotrope; If FAM passage does not have Cq value reading, then represent that this sample does not carry-α ^21.9allelotrope.Result as described in Table 4.

Table 4:

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

Result shows, and wild-type sample does not detect Cq value, and positive sample detects Cq value, detects an example-α in random sample ^21.9allelotrope.

Claims (2)

1. based on the rapid detection-α of fluorescent hydrolysis probe method ^21.9the allelic test kit of absence type thalassemia, comprises amplimer and fluorescent probe, it is characterized in that:

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, wherein:

21.9-F：5’-GGAGCTTTTCCTTCCCTGGAACG-3’；

21.9-R：5’-TGTGGTTGGAGAATGGAGGTGG-3’；

Described fluorescent probe is the fluorescent probe 21.9-Prob of specific detection 21.9-F and 21.9-R amplified production, is specially:

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

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