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PCR amplification primer set, amplification reagent and kit for rapidly detecting Thailand alpha-thalassemia

A technology for thalassemia and amplification primers, applied in the field of kits for single-tube direct PCR detection of Thai-type α-thalassemia

Inactive Publication Date: 2019-01-01
陈治中
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, there is no patent for direct detection of thalassemia using whole blood, amniotic fluid and DBS, and there is no such product on the market

Method used

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  • PCR amplification primer set, amplification reagent and kit for rapidly detecting Thailand alpha-thalassemia
  • PCR amplification primer set, amplification reagent and kit for rapidly detecting Thailand alpha-thalassemia
  • PCR amplification primer set, amplification reagent and kit for rapidly detecting Thailand alpha-thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: The detection result of using the kit of the present invention in whole blood samples with known genotypes

[0083] 1. The composition of the kit:

[0084] Can amplify the alpha-globin gene cluster -- THAI The primer set of the characteristic sequence of allele and normal gene (NG_000006.1)

[0085] THAIF, THAIR, APF and APR:

[0086] Table 2 Detection-- THAI Primer sets for thalassemia-characteristic sequences

[0087]

[0088] Preferably, the PCR reaction system is prepared according to the following Table 3 (primer working concentration is 10 μmol / L, MightyAmp DNAPolymerase is 1.25U / μL):

[0089] Using the orthogonal test method, through a large number of experimental comparisons, and through a large number of experimental comparisons, the optimal PCR reaction solution formula and system are finally determined, as shown in Table 3:

[0090] 18 μl of PCR reaction solution, 2 μl of samples such as peripheral blood (villi, amniotic fluid or umbilic...

Embodiment 2

[0100] Example 2: The detection effect of the kit of the present invention in DBS samples.

[0101] 1. The composition of the kit:

[0102] With embodiment 1.

[0103] Preferably, the PCR reaction system is prepared according to Table 3:

[0104] Using the orthogonal test method, through a large number of experimental comparisons, the optimal PCR reaction solution formulation system is finally determined in Table 3: PCR reaction solution 18 μl, DBS sample 1-3 pieces (diameter 1-2mm) (replenish water 2 μl), the total reaction volume was 20 μl.

[0105] 2. Implementation method:

[0106] With embodiment 1.

[0107] 3. Sample source:

[0108] All samples were derived from anticoagulated peripheral blood samples whose genotypes were determined by conventional Gap-PCR technology, and were collected and prepared through filter paper dried blood spot samples. A double-blind experiment was used for detection. Cut a small disc (diameter: 1-2 mm) in the sample area with a punch. ...

Embodiment 3

[0111] Example 3: The detection effect of the kit of the present invention on amniotic fluid samples (or cultured amniotic fluid samples).

[0112] 1. The composition of the kit:

[0113] With embodiment 1.

[0114] Prepare the PCR reaction system according to Table 3:

[0115] 2. Implementation method:

[0116] With embodiment 1.

[0117] 3. Sample source:

[0118] All samples were derived from amniotic fluid samples whose genotypes were determined by conventional Gap-PCR technique.

[0119] 4. Data analysis and result judgment: the same as in Example 1.

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Abstract

The invention relates to a PCR amplification primer set, an amplification reagent and a kit for rapidly detecting a Thailand alpha-thalassemia, and belongs to a molecular diagnosis technology. According to the present invention, the application of the detection kit of the present invention to detect --<THAI>-deletion thalassemia only consumes 2 h; the detection result of the detection kit of the present invention is consistent with the detection result of the conventional Gap-PCR method using DNA extraction, wherein the conventional Gap-PCR method using DNA extraction requires more than 4 h, and further has disadvantages of high cost, cumbersome operation and easy contamination and degradation of DNA while the detection of --<THAI>-deletion thalassemia with the Direct PCR method has advantages of rapidness, sensitivity, safety and the like, such that the kit of the present invention can be used for detecting --<THAI> deletion thalassemia.

Description

technical field [0001] The invention relates to a molecular diagnosis technique, in particular to a single-tube direct PCR detection kit for Thai type α-thalassemia. Background technique [0002] Thalassemia (Thalassemia, referred to as thalassemia) is one of the most common single-gene genetic diseases in the world. It is caused by a defect in the globin gene; resulting in reduced or missing globin chain synthesis, resulting in an imbalance in the α-chain / β-chain ratio that forms hemoglobin. A group of disabling and fatal genetic hemolytic protein diseases that seriously threaten human health. It is conservatively estimated that there are nearly 200 million thalassemia gene carriers in the world, and the affected individuals are mainly from the Mediterranean region, the Middle East, Transcaucasus, Central Asia, the Indian subcontinent, Southeast Asia and East Asia, and southern China, including Guangxi, Guangdong, and Hainan in southern my country. and other provinces. [...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q2600/16C12Q2600/166
Inventor 陈治中
Owner 陈治中
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