Primers, probes and kit for detecting gene mutation caused by G6PD deficiency disease

Abstract

The invention relates to primers for detecting gene mutation caused by G6PD deficiency disease. The primers are as shown in SEQ ID NO: 1-32. The invention further relates to probes for detecting gene mutation caused by G6PD deficiency disease. The probes are as shown in the sequences SEQ ID NO: 33-67. The invention further relates to a kit for detecting gene mutation caused by G6PD deficiency disease. The kit comprises a PCR reaction liquid I, a PCR reaction liquid II and a PCR reaction liquid III. The kit for detecting gene mutation caused by G6PD deficiency disease provided by the invention has the advantages of high coverage rate, high detection rate of female heterozygote, low cost and high accuracy.

Description

technical field [0001] The invention relates to gene detection technology, in particular to primers, probes and kits for detecting G6PD deficiency gene mutation. Background technique [0002] Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is one of the most common human enzyme deficiency diseases. The essence of G6PD deficiency is the G6PD gene mutation, which is an X-linked incomplete dominant inheritance. The clinical manifestations of patients vary. If G6PD deficiency can be detected and given early intervention, it can often play a very good preventive effect. Because males have only one X chromosome, there are only two types of hemizygotes, normal and significantly deficient. Females have two X chromosomes, and they are divided into normal, heterozygous and homozygous groups according to the number of G6PD mutation genes they carry. Heterozygotes account for the vast majority of female patients. It has long been confirmed at home and abroad that G6P...

AI Technical Summary

Problems solved by technology

Generally speaking, although the detection rate of heterozygotes by the above-mentioned molecular biology methods is as high as 90% to 99%, the technique is complicated, expensive, and time-consuming. It is only used for scientific research and has not been widely used in clinical hospitals. Therefore, It is a basic clinical need to develop a simple, fast, and inexpensive genetic detection and screening method to increase the detection rate of heterozygotes

[0004] So far, more than 160 G6PD gene mutations have been reported in the world, and at least 34 have been found in the Chinese population. G1376T, G1388A, and A95G are the three most common mutations in Chinese people. There is no G6PD deficiency that is maturely used in clinical practice on the market. Gene diagnosis products, technical methods used in laboratory scientific research, such as ARMS, restriction endonuclease method, HRM and DHPLC method, etc., all have the disadvantage of being difficult to promote in clinical application

[0005] Although the existing molecular detection methods can better solve the problem of heterozygous detection in women with G6PD deficiency, different methods have their own limitations: (1) DHPLC requires high equipment and poor applicability; (2) The throughput of ARMS detection is very limited, and PCR post-processing is required, which is prone to contamination and cause false positive results; (3) multiple allele specific PCR (multiplex allele specific PCR, MAS-PCR) method is relatively simple, economical and reliable , but in a reaction system, multiple pairs of primers tend to interfere with each other and affect the PCR results, so a PCR reaction tube is only suitable for detecting a limited number of mutations. For the diagnosis of G6PD deficiency so many mutations, the detection The output rate is limited; (4) Although the fluorescent PCR melting curve method has high sensitivity, it has low accuracy and low throughput, and it is difficult to interpret the results of multiple sites on the same tube, especially the melting front of heterozygotes is more complicated and difficult to interpret , the clinical application is limited; (5) The reverse dot hybridization method is sensitive, reliable, and low in cost, but the operation is cumbersome, and PCR products need to be post-processed, which is easy to cause contamination, and the result interpretation is easily affected by subjective

(6) The sequencing method has many steps, requires special expensive instruments, and the detection cost is expensive

[0006] The defects of the above methods directly limit their clinical application in G6PD deficiency gene detection, that is, there is a problem that clinical application is difficult to promote; in addition, the gene detection kits developed based on the above methods also have the defect of low coverage

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