Primer sequence and kit for detecting glucose-6-phosphate dehydrogenase (G6PD) gene mutation

Abstract

The invention discloses a primer sequence and kit for detecting pathogenic mutation related to glucose-6-phosphate dehydrogenase (G6PD) deficiency. The kit comprises five pairs of amplification primers, 16 single-base extension primers and a special reagent for pretreatment and detection, wherein the amplification primers can specifically amplify 16 common mutation site regions of G6PD genes related to G6PD deficiency; and the 16 single-base extension primers are used for detecting genotypes of 16 mutation sites of the G6PD genes. By the kit, one-hole detection of 16 common mutations related to clinical G6PD deficiency pathopoiesis can be realized; and the kit is high in sensitivity, specificity and accuracy, simple and convenient to operate, low in cost, high in throughput, quick in detection, automatic in result interpretation and easy for clinical popularization and application. The primer sequence and kit can be applied to prenatal diagnosis or neonatal hereditary disease screeningdetection, and a reliable detection reagent is provided for quick detection of G6PD deficiency carriers or patients.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and specifically relates to a primer sequence and a kit for detecting glucose-6-phosphate dehydrogenase (G6PD) gene mutations based on a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) platform . Background technique [0002] Glucose-6-phosphate dehydrogenase deficiency (G6PDd) is caused by the decrease of G6PD activity due to the mutation of the gene regulating glucose-6-phosphate dehydrogenase (G6PD). It is an X-linked incomplete dominant genetic disease. Its disease is often induced by eating broad beans by mistake, so it is also called "bava bean disease". Under conditions such as infection, oxidative stress, food or drug-induced, patients may develop acute hemolytic anemia and hyperbilirubinemia, and severe cases may cause kernicterus, which may even be life-threatening. Prevention of this disease is important, and G6PDd patients can be...

AI Technical Summary

Problems solved by technology

Fluorescence quantification combined with melting curve method has limited single-well detection throughput, and can only detect known mutations at a small number of sites. Specific primers need to be designed for each mutant genotype at each site; multi-hole and multiplex per well is required to cover common sites. Detection, multiple probe design requirements are high, the technology is difficult, the cost of detection probes is high, and the results are interpreted manually, which is prone to misjudgments; the reverse dot hybridization system needs to design probes for each locus and each genotype, and the cost High, the resolution depends on the effectiveness of the probe, it is prone to non-specificity, and the detection process is long, the operation is complicated, the system is open, and the samples are easily contaminated; the Sanger sequencing method is the gold standard for sequencing, but the process is long, the operation is complicated, and requires Manual interpretation of each site, covering common sites requires multi-hole detection, high cost, and low throughput; although high-throughput sequencing can detect all pathogenic sites of G6PD at one time, the cost of instruments and detection reagents is high, and detection When the number of samples is small, it will cause waste, resulting in higher costs, and the system requires a high amount of nucleic acid samples (above 50ng), and newborn heel blood samples are small in size and low in concentration, often requiring screening and testing of multiple items. Therefore, it is difficult to obtain enough nucleic acid templates; the high-throughput sequencing detection process is as long as 3-5 days, the operation is complicated, and professional bioinformatics analysts are required to analyze the data, and the results are difficult to interpret, so it cannot be used as a screening item at all levels of the country Large-scale promotion and application in local small and medium-sized hospitals

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