Primer sets and kits for detection of deletional α-thalassemia

A technology of thalassemia and primer combination, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of unstable amplification system and high GC content in the α-globin gene region, and achieve Single amplification band, good compatibility, excellent sensitivity and specificity

Active Publication Date: 2022-02-15
重庆浦洛通基因医学研究院有限公司
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Problems solved by technology

[0010] In order to solve the problem of unstable amplification system due to high GC content in the α-globin gene region, a stable detection-α 3.7 and -(α) 20.5 Deletion Primer Combinations

Method used

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  • Primer sets and kits for detection of deletional α-thalassemia
  • Primer sets and kits for detection of deletional α-thalassemia
  • Primer sets and kits for detection of deletional α-thalassemia

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Experimental program
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Effect test

Embodiment 1

[0069] Embodiment 1 adopts the present invention to be used for stable detection-α 3.7 and -(α) 20.5

[0070] 1. Detection-alpha 3.7 and -(α) 20.5 Primer combination

[0071] The study found that -α 3.7 and -(α) 20.5 The GC content in the region is high, and it is easy to form a complex secondary structure to hinder the effective amplification of the product. The results of amplification of this region based on conventional primer design are not ideal, and the purity of the template is high, and the amplification results are sometimes absent, which is likely to cause missed detection and pose a huge risk to clinical diagnosis.

[0072] The present invention is to realize single-tube multiplex PCR amplification and simultaneously detect-α 3.7 and -(α) 20.5 Two genotypes, especially to avoid the problem that the high GC content region is easy to form a secondary structure that hinders the effective amplification of specific products, we have conducted a large number of e...

Embodiment 2

[0096] Example 2 The primer combination and kit of the present invention detect 7 kinds of deletion thalassemia

[0097] In order to achieve higher primer amplification efficiency, this program also selects a batch of different types of PCR stabilizers and enhancers for screening and optimization, in order to increase the yield of desired PCR products or reduce non-specific products. Various categories of PCR enhancers can be roughly divided into the following categories according to their mechanism of action: (1) co-solvents used to amplify GC-rich and complex secondary structure templates; (2) stabilizers used to protect DNA polymerase activity; ( 3) Secondary enhancers for optimizing primer and template binding.

[0098] Common stabilizers and enhancers include 1% to 5% DMSO (dimethyl sulfoxide), 1% to 5% glycerin, 1.25% to 10% (v / v) formamide, 0.5mM to 2mM betaine, 0.01 %~0.1% (w / v) BSA (bovine serum albumin), 0.1M~1M trehalose, etc. This program mainly selects different...

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Abstract

The invention relates to in vitro molecular diagnosis technology, in particular to a primer combination and a kit for detecting deletion type α-thalassemia. The primer combination can stably detect -α 3.7 and-(α) 20.5 The two deletion types overcome the problems of high GC content in the α-globin gene region and unstable amplification system; at the same time, the annealing temperature of each primer is within the same temperature range, which avoids non-specific amplification caused by different annealing temperatures. The phenomenon that the amplification efficiency varies. In addition, the primers have good compatibility and no dimer is produced. The kit overcomes the influence between primers, and can detect 7 genotypes of thalassemia in a single tube and multiplex. The need for rapid and convenient clinical detection of thalassemia creates conditions for a more comprehensive screening of thalassemia, and provides a scientific basis for premarital and prenatal examinations and the diagnosis of thalassemia in fetuses during pregnancy.

Description

technical field [0001] The invention relates to an in vitro molecular diagnosis technique, in particular to a combination of primers and a kit for detecting deletion type α-thalassemia. Background technique [0002] Thalassemia (Thalassemia, referred to as thalassemia) is a widely prevalent hereditary hemolytic disease in the world. It is due to the loss or defect of the globin gene, which leads to the loss or reduction of the synthesis of the globin chain, which makes the ratio of the α / β chain forming hemoglobin unbalanced. , causing a group of disabling and fatal genetic hemolytic protein diseases that seriously threaten human health, and it is also one of the largest single-gene genetic diseases in the world. There are at least 350 million people carrying this gene in the world, mainly distributed in countries along the Mediterranean Sea and Southeast Asian countries. It is more common in southern my country, and it is one of the genetic diseases with the highest incide...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2565/125C12Q2545/113
Inventor 罗锋韩勋领孙松松张军谢海迎
Owner 重庆浦洛通基因医学研究院有限公司
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