Primer group and kit for detecting mutant and deletion genes of thalassemia and application of primer group and kit

Abstract

The invention discloses a primer group and a kit for detecting mutant and deletion genes of thalassemia and application of the primer group and the kit. The primer group comprises primers as shown inSEQ ID NO.1-SEQ ID NO.32. The kit comprises the primer group. The primers are designed for exons, regulation regions and transcription regions of three genes HBA1, HBA2 and HBB, primer amplification regions comprise common deletion sites and mutation sites and more than 500 sites including new mutation sites, and the mutation number detected by the invention is far higher than that detected by existing detection methods. Meanwhile, the detection difference of deletion thalassemia and mutant thalassemia is overcome, the deletion thalassemia and the mutant thalassemia are amplified at the same time in the same PCR reaction, known mutation sites and hotspot sites can be detected, new mutation sites can be found too, the operation is easy, and the reagent cost is reduced.

Description

technical field [0001] The invention relates to the technical field of thalassemia detection, in particular to a primer set, a kit and an application thereof for detecting thalassemia mutant and deletion genes. Background technique [0002] Thalassemia (thalassemia, referred to as thalassemia) is a group of serious threats to human health caused by a defect in the globin gene, resulting in reduced or absent globin chain synthesis, resulting in an imbalance in the α-chain / α-chain ratio that forms hemoglobin. The lethal and disabling hereditary blood diseases are a group of single-gene genetic diseases that are the most common in the world and have the greatest impact on human health. They are also genetic diseases recommended by the World Health Organization for priority prevention. They mainly occur in countries along the Mediterranean Sea, Southeast Asia, In Africa and southern China, it is conservatively estimated that there are nearly 200 million thalassemia gene carriers i...

AI Technical Summary

Problems solved by technology

[0003] The methods for thalassemia detection mainly include: Gap-PCR (PCR across breakpoints), PCR-RDB (PCR-reverse dot hybridization), real-time fluorescent quantitative PCR, PCR-ASO (PCR reaction combined with oligonucleotide probes for Dot hybridization), microarray chip method; these methods are generally simple to operate and low in cost, but Gap-PCR can only be used to detect deletion thalassemia, and PCR-RDB, real-time fluorescent quantitative PCR, PCR-ASO and other methods can only be used Detects 17-20 known mutations

[0004] The PCR+sequencing method (PCR-sequencing) has the advantages of detecting multiple mutation types, accurate results, and the ability to discover new mutation types. The disadvantage is that the detection cost is high and corresponding equipment is required

It has been reported that the high-throughput sequencing method is used to detect thalassemia, and the primers are also divided into 2-5 primer sets during PCR amplification. In this way, 2-5 PCR reactions are required to complete the detection of deletion and mutation sites in a sample , increase the cost of reagents, and the operation is cumbersome

[0005] The current conventional detection technology is gap-PCR technology, which has the following obvious technical bottlenecks: First, this method must use a pair of primers and a PCR reaction to detect a molecular diagnosis of a deletion type. Qualitative detection one by one. Therefore, for unknown new deletion types, it is necessary to cause missed detection; moreover, more than 30 types of deletions have been found so far, and it is not realistic to use more than 30 PCR reactions to detect a specimen. Detect several common deletion types in this area. If the phenotype of the tested individual is normal and these common deletion types are not detected, no further testing will be done and a negative result will be reported. In this way, those rare deletions will inevitably be missed. A carrier of static α-thalassemia caused by the deletion type of α-globin gene will have a 1 / 4 probability of giving birth to a child with HbH when mated with a carrier of standard α-thalassemia. is a serious public health problem

Second, due to the high GC content of the α-globin gene cluster and the structural specificity of highly homologous genes, and the deletion range and length of each deletion type are different, and the breakpoint of some deletion types is unknown, etc. , resulting in different PCR amplification systems and amplification conditions for various types of deletions. Some PCR reactions involve long fragments and high GC content, and special Taq enzymes and PCR additives are required. In some cases, due to the use of a special amplification system, it is even difficult to ensure the effective amplification of PCR, which may lead to false negatives; and, because the reaction systems and reaction conditions of different deletion types are different Therefore, it is difficult to design a suitable multiplex PCR system to simultaneously amplify and detect multiple deletion types; therefore, this qualitative molecular diagnostic approach is difficult to detect, high in cost, cumbersome to operate, low in detection throughput, and difficult to realize automation and standardization

[0006] At the same time, due to the differences in the detection of deletion-type thalassemia and mutant-type thalassemia, it is difficult for existing technical solutions to simultaneously amplify deletion-type and mutant-type in the same PCR reaction, and only detect known mutations points and hotspots, no new mutations can be found

Images