Primer probe and kit for detecting copy number of [alpha] globin gene by using microdroplet digital PCR (Polymerase Chain Reaction), and application of primer probe and kit

Abstract

The invention discloses a primer probe and a kit for detecting the copy number of an [alpha] globin gene by using microdroplet digital PCR (Polymerase Chain Reaction), and application of the primer probe and the kit. The primer probe is as shown in SEQ ID NO.1 to SEQ ID NO.4. According to the invention, all [alpha] globin gene copy number deletion and repetition types can be detected by using a pair of primers and two probes, and the generation of a relatively high effective droplet number can be ensured, so that the accuracy and the stability of a result are ensured. According to the method, cell culture is not needed, and the required sample size is small; the detection period is short, and the whole detection process can be completed within 4 hours; the automation degree is high, repeatability is good, and operation is easy; and the kit has the characteristics of high sensitivity, high accuracy, high flux and the like.

Description

technical field [0001] The invention relates to a method for detecting the copy number of α-globin, in particular to a primer probe, a kit and an application for detecting the copy number of α-globin gene by microdroplet digital PCR. Background technique [0002] Thalassemia is the genetic disease with the highest incidence and the greatest impact in the southern provinces of my country. The gene carrier rate of α-thalassemia among the population of childbearing age in our province is 11.31%, and that of β-thalassemia is 4.53%. α-globin gene copy number variation includes α-gene duplication and α-gene deletion. The clinical phenotype of thalassemia is closely related to α-globin gene copy number. When mutated, it causes beta-thalassaemia intermedia. The vast majority of α-thalassemia is caused by the deletion of the α gene. At present, there are more than 30 types of α gene deletions reported. However, the kits currently on the market usually only detect 3 types of deletion...

AI Technical Summary

Problems solved by technology

The cost of multiplex PCR method is low, and the requirements for equipment are not high, but it is limited to the detection of specific duplication or deletion types. The type of α globin gene copy number variation is complex and diverse, and multiple gap-PCR method will inevitably lead to missed diagnosis.

MLPA is cumbersome to operate, takes a long time, requires high equipment requirements, and is costly. It is mainly used for the detection of copy number variation and genotype confirmation of unknown types in individual cases, and is not suitable for large-scale application as a molecular screening method.

Fluorescent quantitative PCR needs to establish a standard curve and Ct value for quantitative analysis of copy number, not direct quantification, and the accuracy of fluorescent quantitative PCR decreases significantly when the copy number is high, which also limits the application of this method

Next-generation sequencing technology has high requirements for equipment and personnel, and high cost. It is currently not suitable for promotion as a first-line clinical screening method.

[0005] In 2013, scholars in Thailand established a microdroplet digital PCR method for detecting Southeast Asian α-deletion, and applied the microdroplet digital PCR method to the gene detection of thalassemia, but this method can only detect one type of deletion, which limits clinical application

In 2016, Malaysian scholars established a method for detecting triplets and deletions by microdroplet digital PCR, which confirmed that microdroplet digital PCR is an accurate and fast method for detecting deletions and triplets, and the detection results are stable and reliable. Four PCR reactions need to be carried out at the same time, which reduces the detection throughput and increases the detection cost to a certain extent

At present, only the team of Professor Zhou Wanjun of Southern Medical University established the application of droplet digital PCR to detect ααα in 2017. anti3.7 method, but this method can only detect one type of triplet, which cannot meet the clinical needs

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