Primer set, chip and kit for genotyping detection of alpha thalassemia and beta thalassemia

A thalassemia, primer set technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as large sample volume, large sample storage space, and easy contamination.

Pending 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

[0009] In addition, these techniques require DNA extraction, which is cumbersome and complicated, time-consuming, prone to cross-contamination, and experiments require DNA automatic extraction instruments, precision instruments and reagent consumables, which are expensive and cannot be widely used clinically.
The quality of DNA is a very critical factor for the success of PCR, and the process of extracting and purifying DNA is easily contaminated, making the experiment prone to false negatives or false positives, which can easily cause missed or false detections
[0010] In addition, at the same time, a large number of samples are required. For the transportation of samples collected in remote areas, cold chain equipment is required, and a three-level packaging system is required, and the storage space for samples is large; the risk factor of biosafety is high.
For samples such as whole blood, amniotic fluid, and DBS, there is no patented technology for direct detection of thalassemia without DNA purification, and there is no such product on the market

Method used

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  • Primer set, chip and kit for genotyping detection of alpha thalassemia and beta thalassemia
  • Primer set, chip and kit for genotyping detection of alpha thalassemia and beta thalassemia
  • Primer set, chip and kit for genotyping detection of alpha thalassemia and beta thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Embodiment 1: Using the kit specimen source and PCR template type of the present invention

[0119] 1.1 Sample collection and preparation of PCR template: a) Sample collection: Specimens are derived from anticoagulated peripheral blood, DBS samples, embryonic villi tissue, amniotic fluid or umbilical cord blood, embryonic genetic material including gametes such as sperm or eggs, cleavage-stage embryos Blastomeres, blastocyst trophectoderm cells, ie, blastocyst cells, etc.; b) Preparation of PCR templates: the above samples can be directly used as templates, or DNA extracted from the above samples by nucleic acid can also be used as templates.

[0120] 1.2 DBS sample preparation

[0121] The above 1.1 filter paper dry blood spot sample (DBS) collection and preparation are as follows:

[0122] ①. Use Whatman903 filter paper (or ordinary filter paper). Mark the subject number and date of collection on the filter paper. ② Both peripheral blood and anticoagulated blood ca...

Embodiment 2

[0124] Example 2. Design of specific primers for PCR amplification and determination of PCR reaction system

[0125] 1.1 Primer design

[0126] The α-globin and β-globin gene sequences were obtained from the GenBank database, and a primer set was designed for PCR amplification according to the mutation regions covered by the α-globin and β-globin genes, and 3 pairs of primer combinations were placed in Perform direct multiplex PCR in the same reaction tube to simultaneously amplify the α-thalassemia non-deletion mutation and the β-thalassemia gene mutation; another primer set is also placed in another reaction tube for direct multiplex PCR amplification of the deletion α-thalassemia; primers were synthesized by a professional biological company. Primer sequences are shown in Table 3,

[0127] Table 3 Specific primers in PCR reaction solution Ⅰ and PCR reaction solution Ⅱ

[0128]

[0129] 1.2 Determination of multiplex PCR reaction system

[0130] Using the orthogonal t...

Embodiment 3

[0151] Example 3: Design, spotting and immobilization of oligonucleotide probes

[0152] 3.1. Design and screening of probes

[0153] The α-globin gene sequence was obtained from the GenBank database, and 7 kinds of α-thalassemia mutations (QS, CS, WS, -- SEA 、-- THAI , -α 3.7 and-alpha 4.2 ) and 19 kinds of oligonucleotide probe combinations for β-thalassaemia gene mutations; and design a chromogenic control probe (CC) (Table 6 and Table 7). Synthesized by a professional biological company.

[0154] Table 6 Region-specific probes for β-thalassemia gene mutation

[0155]

[0156]

[0157] Table 7 Region-specific probes for α-thalassemia gene mutation

[0158]

[0159]

[0160] 3.2. Preparation of gene chip.

[0161] The preparation method of the gene chip of the present invention comprises the following steps:

[0162] (1) Preparation of the working solution of the oligonucleotide probe: use the probe diluent (0.5M Na 2 CO 3 and 0.5M NaHCO 3 solution) we...

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PUM

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Abstract

The present invention provides a primer set for detecting alpha thalassemia and beta thalassemia, and belongs to the field of molecular biology, wherein the primer set comprises an alpha primer set and a beta primer set. The invention further provides a chip capable of simultaneously detecting alpha thalassemia and beta thalassemia, wherein probes having sequences represented by SEQ ID NO:1-31 areimmobilized on the chip. Based on the primer set and the chip, the present invention further provides a kit capable of simultaneously detecting alpha thalassemia and beta thalassemia. The kit of theinvention has advantages of short detection time, low cost, convenient operation, closed tube operation and pollution reducing, and has great significance in the screening of thalassemia population, the genetic counseling and the prenatal diagnosis.

Description

technical field [0001] The invention belongs to the field of molecular biology medicine, and relates to a technology for detecting α and β thalassemia, in particular to a direct PCR (Direct PCR) combined with hybridization technology for simultaneously detecting 7 kinds of α thalassemia mutations and 19 kinds of point mutations β thalassemias kit. Background technique [0002] Thalassemia (thalassemia, referred to as thalassemia), is due to α or β globin gene deletion or mutation leads to globin chain synthesis disorder, resulting in an imbalance in the ratio of α chain to β chain, and the relative excess chain is in a free state, which leads to Hemoglobin is unstable, redundant globin chains are deposited on the red blood cell membrane, prone to oxidative denaturation, and the permeability and fragility of the red blood cell membrane are changed, leading to hemolytic anemia. Clinically, chronic progressive hemolysis with varying symptoms is often manifested sexual anemia. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q2600/16C12Q2600/166
Inventor 陈治中卿吉琳
Owner 陈治中
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