Nucleic acid membrane strip and reagent kit for non-deletion type alpha-thalassemia gene detection and detection method

A thalassemia and non-deletion technology, applied in the field of non-deletion α-thalassemia gene detection, can solve the problems of increasing the risk of missed detection and non-inclusion, and achieve high specificity, simple operation, and increase amplification efficiency. Effect

Inactive Publication Date: 2015-10-21
深圳益生堂生物企业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the amplified fragment used for the detection of non-deleted α-thalassaemia gene is also 1.8kb, which has the disadvantage that the labeled amplified fragment is too long; in addition, the non-deleted α-thalassemia gene detection probe does not contain the most Common non-deletion thalassemia gene alpha WS Alpha / detection probes, increasing the risk of missed detections

Method used

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  • Nucleic acid membrane strip and reagent kit for non-deletion type alpha-thalassemia gene detection and detection method
  • Nucleic acid membrane strip and reagent kit for non-deletion type alpha-thalassemia gene detection and detection method
  • Nucleic acid membrane strip and reagent kit for non-deletion type alpha-thalassemia gene detection and detection method

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Experimental program
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Effect test

preparation example Construction

[0066] 1.2 Preparation of nucleic acid membrane strips

[0067] Probe preparation: Dilute the probes with 0.5mol / L carbonate buffer (pH8.4) to the sample concentration, mix well, arrange them in order, and put them in an ice box for later use.

[0068] Membrane strip pre-treatment: Cut the membrane into a size suitable for printing by the printer, and use the printer to print the spotting area as shown in Table 2. Table 2 is the position table of the nucleic acid membrane strip.

[0069] Table 2

[0070] WSN

WSM

QSN

QSM

CSM

CSN

[0071]Soak the membrane with 0.1mol / L HCl solution twice, 5 minutes each time, then soak the membrane with 16% EDAC solution for 15 minutes, and finally wash it with purified water 4 times, 5 minutes each time, and place it at room temperature for more than 30 minutes.

[0072] Spotting the membrane: spot the probes on the corresponding area according to the position of the probes on the membrane, 0.8 μl for each ...

Embodiment 1

[0091] Preparation of sample genomic DNA

[0092] This kit has no specified requirements for the extraction method of human genomic DNA. Traditional methods such as phenol-chloroform method or commercial nucleic acid extraction kits can be used to extract human genomic DNA. It is recommended to use the blood / cell / tissue of Tiangen Biochemical Technology (Beijing) Co., Ltd. Genomic DNA extraction kit, and operate according to the instructions of the kit. Blood samples should be anticoagulated with EDTA or sodium citrate instead of heparin.

[0093] 2. Preparation of nucleic acid membrane strips

[0094] 2.1 Probe preparation

[0095] Dilute the probes with 0.5mol / L carbonate buffer (pH8.4) to the sample concentration, mix well, arrange them in order, and put them in an ice box for later use. The sample concentration of the probe is shown in Table 6. Table 6 is the sample concentration table of probes.

[0096] Table 6

[0097]

[0098] 2.2 Pretreatment of film strips: ...

Embodiment 2

[0117] The hybridization membrane strip and the kit of the present invention detect clinical sample results and compare the gold standard sequencing results.

[0118] Select 1248 cases of clinical samples, respectively adopt the hybridization membrane strip and kit of the present invention to detect the mutations of WS, QS, and CS genes in the samples, and detect the α2 genes CD30 (-GAG), CD31 (AGG>AAG), CD43 / 44(-C), CD49(-CG), CD59(GGC>GAC) and WS, QS, CS gene mutations, the detection results are shown in Table 8, with the sequencing results as the standard, the hybridization membrane strip and kit of the present invention detect clinical samples The positive and negative results of the non-deleted α-thalassemia gene are shown in Table 9. Table 8 is a comparison table of the detection results of the hybridization membrane strip and the kit of the present invention and the dideoxy chain termination sequencing method.

[0119] Table 8

[0120]

[0121]

[0122] Table 9...

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Abstract

The invention relates to a nucleic acid membrane strip for non-deletion type alpha-thalassemia gene detection. The nucleic acid membrane strip comprises a substrate and specific oligonucleotide probes fixed to the substrate, wherein the sequences of the specific oligonucleotide probes are shown in SEQ ID NO: 1-6. The invention further relates to a reagent kit for non-deletion type alpha-thalassemia gene detection and a detection method. The nucleic acid membrane strip for non-deletion type alpha-thalassemia gene detection has the advantages of being high in adaptability and specificity. The reagent kit for non-deletion type alpha-thalassemia gene detection has the advantages of being high in amplification efficiency, high in probe and hybridization template combination efficiency, higher in detection sensitivity and better in specificity and accuracy.

Description

technical field [0001] The invention relates to gene detection technology, in particular to a nucleic acid membrane strip, a kit and a detection method for non-deletion α-thalassemia gene detection. Background technique [0002] Thalassemia is a group of hemolytic anemias, showing the law of autosomal recessive inheritance, mainly caused by defects such as deletion and mutation of α-globin gene or β-globin gene, which cause the inhibition of the synthesis of the corresponding globin chain or the loss of its function due to. The disease is more common in Guangxi, Guangdong, Hainan, Sichuan and Guizhou provinces in southern my country, and can be divided into two types: α-thalassemia and β-thalassemia. [0003] α-thalassemia is mainly caused by large fragment deletion of α-globin gene, and a small part is caused by point mutation of α-globin gene. α-thalassemia caused by point mutation is called non-deletion α-thalassemia. At least 12 non-deletion α-thalassemia gene mutation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/156
Inventor 陈立炎张文
Owner 深圳益生堂生物企业有限公司
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