Multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting glucose-6-phosphate dehydrogenase deficiency G6PD (Glucose-6-phosphate dehydrogenase) gene variation sites

Abstract

The invention relates to multiple fluorescent quantitative PCR (Polymerase Chain Reaction) primers, a probe and a kit for detecting glucose-6-phosphate dehydrogenase deficiency G6PD (Glucose-6-phosphate dehydrogenase) gene variation sites. The invention provides three G6PD gene mutation sites found for the first time, and the three mutation sites are respectively a G6PD gene c.521Ggt, a G6PD gene c.521Ggt and a G6PD gene c.521Ggt. C, c, 661 Tgt, 661 Tgt; c, c, 680Ggt; c variation sites expand the pathogenic variation spectrum of the G6PD gene. The invention further provides a primer, a probe composition and a kit for detecting the G6PD gene variation site of the glucose-6-phosphate dehydrogenase deficiency disease. The primer, the probe composition and the kit are used for screening or diagnosing the glucose-6-phosphate dehydrogenase deficiency disease. The sequences of the primer and the probe are as shown in SEQ ID NO.1-10. The kit disclosed by the invention can comprehensively cover three new pathogenic variation sites of the G6PD gene related to the glucose-6-phosphate dehydrogenase deficiency, is high in accuracy and can specifically detect the pathogenic variation sites.

Description

Technical Field

[0001] The present invention relates to the technical field of gene diagnosis, and in particular to a primer and probe combination and a kit for detecting a variation site of a glucose-6-phosphate dehydrogenase deficiency G6PD gene. Background Art

[0002] Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency, OMIM: 305900) is a genetic metabolic disease and one of the most common X-linked genetic diseases in humans. It is an X-linked incomplete dominant inheritance. The disease is caused by a mutation in the G6PD gene, which leads to a decrease in the activity of the G6PD enzyme in red blood cells, thereby weakening the resistance of red blood cells to oxidative stress and making hemolytic anemia more likely to occur. The clinical manifestations of patients range from asymptomatic to acute hemolytic anemia (paroxysmal hemolysis) induced by infection, drugs or food, and even spontaneous chronic hemolytic anemia (chronic non-spherocytic hemolytic anemia). The most serious manifestation is neonatal hyperbilirubinemia and kernicterus, and the manifestations vary greatly.

[0003] G6PD is an enzyme located in red blood cells. It mainly participates in the pentose phosphate pathway and catalyzes the dehydrogenation reaction of 6-phosphoglucose to generate 6-phosphogluconic acid and reduced nicotinamide adenine dinucleotide (NADPH). NADPH converts oxidized glutathione (GSSG) into reduced glutathione (GSH) under the catalysis of glutathione reductase. As an important antioxidant in cells, the latter can remove hydrogen peroxide in cells and protect cells from oxidative damage. In patients with G6PD deficiency, due to the reduced synthesis of NADPH, the level of GSH in cells decreases, causing red blood cells to deform and rupture easily under oxidative stress, thereby causing hemolysis.

[0004] The clinical manifestation of G6PD deficiency is mostly hemolytic anemia, which is easily triggered when exposed to oxidative substances (such as broad beans, certain drugs, infections, etc.). The favine component in broad beans can trigger an oxidative reaction, causing red blood cell damage, which is why the disease is named "favism". In addition, severe hyperbilirubinemia may occur in the neonatal period of G6PD deficiency, and even cause kernicterus, affecting the development of the nervous system.

[0005] The G6PD gene is located in the Xq28 region of the X chromosome, is about 20kb long, contains 13 exons and 12 introns, and encodes 515 amino acids. G6PD deficiency is an X-linked recessive genetic disease, which is more common in male patients because males only have one X chromosome, and their lack of G6PD enzyme activity cannot be compensated by another normal X chromosome. Female patients are usually heterozygous, and the enzyme activity is affected by mutations, etc., which manifests as varying degrees of reduced enzyme activity.

[0006] The diagnosis of G6PD deficiency mainly relies on genetic testing and enzyme activity determination. Enzyme activity determination methods (such as methemoglobin reduction test, fluorescent spot method, nitro blue tetrazolium method, etc.) can measure the G6PD enzyme activity in red blood cells, but they are affected by many factors and cannot completely and accurately diagnose all types of deficiency. As an accurate means of diagnosis, genetic testing can identify the patient's G6PD gene mutation type and provide important information for clinical treatment and genetic counseling. Different genotypes can determine the degree of enzyme deficiency and prognosis of patients with G6PD deficiency, and reduce the occurrence of adverse medical outcomes by effectively avoiding the intake of toxic drugs and foods (such as broad beans).

[0007] At present, the common methods for detecting G6PD deficiency mutations mainly include enzyme activity assays, gene sequencing, and specific PCR detection of common mutations. Although these methods can meet clinical needs in most cases, they still have significant limitations when detecting multiple mutations at the same time. Traditional G6PD gene sequencing methods such as gene sequencing are complex to operate and costly, and are also limited in sensitivity, specificity, and rapid diagnosis. Especially when faced with rare or newly discovered mutations, they are often limited by high-throughput detection requirements or cost issues. Therefore, there is an urgent need for a detection method that can quickly, efficiently, and accurately identify multiple mutations associated with G6PD deficiency, especially newly discovered mutations, to meet the clinical demand for efficient and variable detection, to improve clinical diagnostic efficiency, and to provide a basis for early intervention. Summary of the invention

[0008] The purpose of the present invention is to provide three G6PD gene mutation sites discovered for the first time to enrich the variation spectrum of the G6PD gene. The three variation sites are c.521G>C, c.661T>C, and c.680G>C variation sites of the G6PD gene. At the same time, the present invention also provides a primer and probe combination for detecting the variation sites of the G6PD gene of glucose-6-phosphate dehydrogenase deficiency and a kit for screening or diagnosing glucose-6-phosphate dehydrogenase deficiency, including specific primers and TaqMan probe combinations for the variation sites of the G6PD gene c.521G>C, c.661T>C, and c.680G>C, respectively.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] The specific primer and probe combination of the present invention is designed using Primer 6.0 software, specifically:

[0011] 1) Targeting the G6PD gene c.521G>C site:

[0012] Forward primer A: 5'-CTCTGATCCTCACTCCCCGAA-3' (SEQ ID NO.1);

[0013] Reverse primer A: 5'-TCACCCTTGTCTGAGTTCTGGA-3' (SEQ ID NO. 2);

[0014] G6PD gene wild-type probe Aw: 5'-AGGTCCCTCCCGAAGGGCTT-3' (SEQ ID NO. 3);

[0015] G6PD gene mutation probe Am: 5'-AGGTCCCTCGCGAAGGGCTT-3' (SEQ ID NO. 4);

[0016] 2) Targeting the G6PD gene c.661T>C site:

[0017] Forward primer B: 5'-ACGAATTCCTCCAGAACTCAGAC-3' (SEQ ID NO. 5);

[0018] Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3' (SEQ ID NO. 6);

[0019] G6PD gene wild-type probe Bw: 5'-ATGGGGCCGAAGATCCTGTTGG-3' (SEQ ID NO. 7);

[0020] G6PD gene mutation probe Bm: 5'-ATGGGGCCGAGGATCCTGTTGG-3' (SEQ ID NO. 8);

[0021] 3) Targeting the G6PD gene c.680G>C site:

[0022] Forward primer B: 5'-ACGAATTCCTCCAGAACTCAGAC-3' (SEQ ID NO. 5);

[0023] Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3' (SEQ ID NO. 6);

[0024] G6PD gene wild-type probe Cw: 5'-GATGTTGTCCCGGTTCCAGATG-3' (SEQ ID NO. 9);

[0025] G6PD gene mutation probe Cm: 5'-GATGTTGTCCGGGTTCCAGATG-3' (SEQ ID NO. 10).

[0026] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0027] In the same reaction system, the 5' ends of the probes respectively carry different reporter fluorescent groups. For example, in reaction system A, the 5' ends of the six probes for detecting three sites are respectively modified with different identifiable fluorescent dyes, including but not limited to one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LCRed640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, LC Red705, etc.

[0028] A detection kit for detecting G6PD gene mutation sites of glucose-6-phosphate dehydrogenase deficiency based on TaqMan probe single-tube multiplex real-time fluorescence quantitative PCR technology, comprising a reaction system A;

[0029] The reaction system A includes the detection primers and detection probes as shown below:

[0030] Detection primers:

[0031] Targeting the G6PD gene c.521G>C site:

[0032] Forward primer A: 5′-CTCTGATCCTCACTCCCCGAA-3′;

[0033] Reverse primer A: 5′-TCACCCTTGTCTGAGTTCTGGA-3′;

[0034] Targeting the G6PD gene c.661T>C site:

[0035] Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′;

[0036] Reverse primer B: 5′-ATCAAGGAAGGACATGGTGATGC-3′;

[0037] Targeting the G6PD gene c.680G>C site:

[0038] Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′;

[0039] Reverse primer B: 5′-ATCAAGGAAGGACATGGTGATGC-3′;

[0040] Detection probe:

[0041] Targeting the G6PD gene c.521G>C site:

[0042] G6PD gene wild-type probe Aw: 5′-AGGTCCCTCCCGAAGGGCTT-3′;

[0043] G6PD gene mutant probe Am: 5′-AGGTCCCTCGCGAAGGGCTT-3′;

[0044] Targeting the G6PD gene c.661T>C site:

[0045] G6PD gene wild-type probe Bw: 5′-ATGGGGCCGAAGATCCTGTTGG-3′;

[0046] G6PD gene mutation probe Bm: 5′-ATGGGGCCGAGGATCCTGTTGG-3′;

[0047] Targeting the G6PD gene c.680G>C site:

[0048] G6PD gene wild-type probe Cw: 5′-GATGTTGTCCCGGTTCCAGATG-3′;

[0049] G6PD gene mutant probe Cm: 5′-GATGTTGTCCGGGTTCCAGATG-3′;

[0050] The 5' end of the probe carries a reporter fluorescent group, and the 3' end carries a quencher fluorescent group; in reaction system A, the 5' end of each probe carries a different reporter fluorescent group, and can be distinguished from each other by a fluorescent quantitative PCR instrument according to different spectral wavelengths;

[0051] The reporter fluorescent group is one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, AlexaFluor 633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LC Red640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, LCRed705, etc., and the quenching fluorescent group is MGB. In the multiplex reaction system, the 5' ends of the probes are respectively provided with different reporter fluorescent groups, and can be distinguished from each other by a fluorescence quantitative PCR instrument according to different spectral wavelengths.

[0052] The reaction system A is 30 μL, and each 30 μL of reaction system A contains:

[0053] 2×premix Taq TM Buffer 15μL, 10μmoL / L Forward primer A 1μL, 10μmoL / L Reverse primer A 1μL, 10μmoL / L Forward primer B 1μL, 10μmoL / L Reverse primer B 1μL, 10μmoL / L probe Aw 1μL, 10μmoL / L probe Am 1μL, 10μmoL / L probe Bw 1μL, 10μmoL / L probe Bm 1μL, 10μmoL / L probe Cw 1μL, 10μmoL / L probe Cm 1μL, template DNA 1μL (≥10ng), 4μL RNase and DNase-free water.

[0054] In the detection kit, when the TaqMan probe is amplified in a single tube multiplex real-time fluorescence quantitative PCR, the amplification reaction procedure is as follows:

[0055]

[0056] The present invention also provides the use of a reagent for detecting a G6PD gene variation site in the preparation of a glucose-6-phosphate dehydrogenase deficiency detection reagent, wherein the G6PD gene variation site comprises at least one of the following sites: a G6PD gene c.521G>C site, a G6PD gene c.661T>C site, and a G6PD gene c.680G>C site;

[0057] The gene number of the wild-type G6PD gene in the NCBI database is: NM_001042351.3,

[0058] Among them, the G6PD gene c.521G>C site is a hemizygous mutation, the 521st base in the G6PD gene coding region mutates from G to C, and the mutation causes the 174th amino acid of the encoded protein to mutate from glycine to alanine;

[0059] The G6PD gene c.661T>C site is a hemizygous mutation, where the 661th base in the G6PD gene coding region mutates from T to C, causing the 221st amino acid in the encoded protein to mutate from phenylalanine to leucine;

[0060] The c.680G>C site of the G6PD gene is a hemizygous mutation. The 680th base in the coding region of the G6PD gene mutates from G to C. The mutation causes the 227th amino acid of the encoded protein to mutate from arginine to proline.

[0061] The G6PD gene c.521G>C site, the G6PD gene c.661T>C site, and the G6PD gene c.680G>C site were discovered by the present invention using high-throughput sequencing technology after excluding other glucose-6-phosphate dehydrogenase deficiency pathogenic genes. These mutations have not yet been included in the gnomAD, HGMD, and ClinVar databases and are pathogenic mutation sites discovered for the first time.

[0062] The reagent for detecting the G6PD gene variation site is one or more of a probe and a primer for detecting the G6PD gene variation site.

[0063] The primers include primers for at least one of the variant sites of the G6PD gene c.521G>C site, the G6PD gene c.661T>C site, and the G6PD gene c.680G>C site, and the primers for the sites are as follows:

[0064] Targeting the G6PD gene c.521G>C site:

[0065] Forward primer A: 5′-CTCTGATCCTCACTCCCCGAA-3′;

[0066] Reverse Primer A: 5'-TCACCCTTGTCTGAGTTCTGGA-3'

[0067] Targeting the G6PD gene c.661T>C site:

[0068] Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′;

[0069] Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3'

[0070] Targeting the G6PD gene c.680G>C site:

[0071] Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′;

[0072] Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3'.

[0073] The probes include probes for at least one variant site among the G6PD gene c.521G>C site, the G6PD gene c.661T>C site, and the G6PD gene c.680G>C site, and the probes for the sites are as follows:

[0074] Targeting the G6PD gene c.521G>C site:

[0075] G6PD gene wild-type probe Aw: 5′-AGGTCCCTCCCGAAGGGCTT-3′;

[0076] G6PD gene mutation probe Am: 5'-AGGTCCCTCGCGAAGGGCTT-3',

[0077] Targeting the G6PD gene c.661T>C site:

[0078] G6PD gene wild-type probe Bw: 5′-ATGGGGCCGAAGATCCTGTTGG-3′;

[0079] G6PD gene mutation probe Bm: 5'-ATGGGGCCGAGGATCCTGTTGG-3',

[0080] Targeting the G6PD gene c.680G>C site:

[0081] G6PD gene wild-type probe Cw: 5′-GATGTTGTCCCGGTTCCAGATG-3′;

[0082] G6PD gene mutation probe Cm: 5'-GATGTTGTCCGGGTTCCAGATG-3'.

[0083] Compared with the prior art, the advantages of the present invention are as follows: the present invention uses high-throughput sequencing technology to exclude other hereditary hemolytic anemia pathogenic genes, and for the first time discovers three pathogenic variation sites in the glucose-6-phosphate dehydrogenase deficiency pathogenic gene G6PD gene, which are: G6PD gene c.521G>C site, G6PD gene c.661T>C site, G6PD gene c.680G>C site, these variation sites are new pathogenic variation related to glucose-6-phosphate dehydrogenase deficiency, and the results are accurate according to the judgment of population frequency, family information, computer bioinformatics analysis, inheritance pattern, clinical phenotype, etc., which expands the pathogenic variation spectrum of the G6PD gene and provides a basis for the diagnosis and genetic counseling of glucose-6-phosphate dehydrogenase deficiency. In addition, the present invention provides a primer and probe composition and a kit for detecting the G6PD gene mutation site of glucose-6-phosphate dehydrogenase deficiency for screening or diagnosing glucose-6-phosphate dehydrogenase deficiency; including specific primers and TaqMan probe compositions for the G6PD gene c.521G>C, c.661T>C, and c.680G>C mutation sites, respectively. Among them, the detection kit and detection method for detecting the G6PD gene mutation site of glucose-6-phosphate dehydrogenase deficiency based on the TaqMan probe single-tube multiple real-time fluorescence quantitative PCR technology of the present invention can comprehensively cover the three new pathogenic mutation sites of the G6PD gene related to glucose-6-phosphate dehydrogenase deficiency. The multiplex TaqMan real-time fluorescence quantitative PCR technology is time-consuming and simple to operate. It only needs to add the DNA specimen of the subject to the reaction system A of the present invention, and then detect it on the machine. There is no need for gel electrophoresis, purification recovery, sequencing and other post-processing. The results are accurate and reliable, which can meet the current clinical urgent need for a simple, fast and accurate patient and prenatal sample diagnostic detection method. In addition, the primers and probes designed by the present invention have strong specificity and can specifically detect the above three pathogenic variant sites discovered for the first time, thereby realizing the diagnosis of glucose-6-phosphate dehydrogenase deficiency and providing a basis for guiding clinical diagnosis and treatment and genetic counseling. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 It is the pedigree chart of proband 1 in Example 2, and the arrow indicates proband 1.

[0085] Figure 2 It is the candidate sequence variation detected by high-throughput sequencing of proband 1 in Example 2.

[0086] Figure 3 This is a BAM file diagram of the high-throughput sequencing results of the c.521G>C variant of the G6PD gene of proband 1 in Example 2.

[0087] Figure 4 It is the pedigree chart of proband 2 in Example 3, and the arrow indicates proband 2.

[0088] Figure 5 It is the candidate sequence variation detected by high-throughput sequencing of proband 2 in Example 3.

[0089] Figure 6 This is a BAM file diagram of the high-throughput sequencing results of the c.661T>C variant of the G6PD gene of proband 2 in Example 3.

[0090] Figure 7 It is the pedigree chart of proband 3 in Example 4, and the arrow indicates proband 3.

[0091] Figure 8 It is the candidate sequence variation detected by high-throughput sequencing of proband 3 in Example 4.

[0092] Fig. 9 This is a BAM file diagram of the high-throughput sequencing results of the c.680G>C variant of the G6PD gene of proband 3 in Example 4.

[0093] Fig.10 It is the real-time fluorescence quantitative PCR result graph of the TaqMan probe of the G6PD gene c.521G>C site in Example 6, wherein the upper left graph is the amplification curve result graph of the c.521G wild type; the upper right graph is the amplification curve result graph of the G6PD gene c.521G>C site heterozygote; the lower left graph is the amplification curve result graph of the c.521G>C site hemizygote.

[0094] Fig.11 It is the real-time fluorescence quantitative PCR result graph of the TaqMan probe of the G6PD gene c.661T>C site in Example 6, wherein the upper left graph is the amplification curve result graph of the c.661T wild type; the upper right graph is the amplification curve result graph of the G6PD gene c.661T>C site heterozygote; and the lower left graph is the amplification curve result graph of the c.661T>C site hemizygote.

[0095] Fig.12 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe of the G6PD gene c.680G>C site in Example 6; wherein, the upper left figure is the amplification curve result diagram of the c.680G wild type; the upper right figure is the amplification curve result diagram of the G6PD gene c.680G>C site heterozygote; the lower left figure is the amplification curve result diagram of the c.680G>C site hemizygote.

[0096] Fig.13 It is the Sanger sequencing result diagram of the c.521G>C site of the G6PD gene in Example 7; wherein the red box is the detection site.

[0097] Fig.14This is the Sanger sequencing result diagram of the G6PD gene c.661T>C site in Example 7, wherein the red box is the detection site.

[0098] Fig.15 It is the Sanger sequencing result diagram of the c.680G>C site of the G6PD gene in Example 7; wherein the red box is the detection site. DETAILED DESCRIPTION

[0099] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0100] Example 1 Detection of pathogenic variants in patients with glucose-6-phosphate dehydrogenase deficiency using the Customized Clinical Exome Capture Kit (TWIST)

[0101] This example uses clinical peripheral blood samples from 238 patients with a clinical diagnosis of glucose-6-phosphate dehydrogenase deficiency and sent to the Furui Medical Laboratory as the research object, and collects clinical symptoms such as skin, liver, spleen, general signs such as body temperature and development, and laboratory tests such as blood routine and glucose-6-phosphate dehydrogenase activity. The proband of the candidate glucose-6-phosphate dehydrogenase deficiency and some family members were tested by the "Customized Clinical Exome Capture Kit (TWIST)" high-throughput sequencing test, which includes the following steps in sequence:

[0102] (1) Sample collection and genomic DNA extraction.

[0103] Blood samples (EDTA anticoagulation) were collected from the proband and / or family members, and genomic DNA from the blood of each family member was extracted according to the instructions of the blood DNA extraction kit (Magen, HiPure Blood & Tissue DNA Kit). The OD260nm / OD280nm of the obtained genomic DNA was between 1.7 and 2.0. The concentration of DNA was measured using Nanodrop one, and the concentration of the obtained genomic DNA was 50-100 ng / μL, and the total amount was 5-10μg. Store at -20℃.

[0104] (2) High-throughput sequencing and bioinformatics analysis of glucose-6-phosphate dehydrogenase deficiency gene mutations.

[0105] First, the extracted genomic DNA was fragmented using the KAPA HyperPlus Library Preparation Kit according to the kit instructions, and the genomic DNA was subjected to enzyme fragmentation, end repair, 3' end A addition, linker connection and PCR amplification; a customized clinical exome capture kit (TWIST) was used to capture the exon region of the Mendelian genetic disease pathogenic gene included in the OMIM database, the intron region of ±20 bp on the side, and other regions with HMGD and ClinVar pathogenic variants. The library was sequenced on a Novasek 6000 sequencer (Illumina, San Diego, CA, USA) (sequencing depth ≥150X, 20X coverage ≥96%). All sequenced samples must pass quality control before data interpretation, and priority should be given to analyzing the pathogenic genes G6PD, EPB41, SPTA1, SPTB, PIEZO1, SLC4A1, HBA1, HBA2, HBZ, HBB, PKLR, CDAN1, CDIN1, KIF23, KLF1, and SEC23B for glucose-6-phosphate dehydrogenase deficiency and other hereditary hemolytic anemia differential diagnosis diseases to ensure that all sequences of these genes can be detected with high quality (20X coverage reaches 100%). If it is found that the 20X coverage of some genes or regions does not reach 100%, additional Sanger sequencing is required to complete the above-mentioned regions to ensure the detection rate of pathogenic variants.

[0106] The secondary and tertiary analysis of sequencing data was completed using the ISoGenetic v1.2.6 system (Shanghai Fujun Gene Biotechnology Co., Ltd.). Specifically, Sentieon BWAv0.7.15 was used to align the NGS sequencing raw data with the human reference genome UCSC NCBI37 / hg19 to obtain the unique alignment sequence aligned to the genome; SAMtools v1.9 and Sentieon GATK software v4.1.4.0 were used to detect and determine the variation in the target region; Remove RunCommonVariants and Remove Global CommonVariants software were used to remove common variations (>5%) in the dbSNP and gnomAD databases; Alamut-Batch standalone version v1.9 was used to analyze the sequence of the target region; and the sequence of the target region was analyzed by the Alamut-Batch standalone version v1.9. The software annotates the variants (annotation databases include: dbSNP, gnomAD, 1000g, ClinVar, OMIM, HGMD Professional Edition, etc.); filterAlamut.py is used to sort the annotated variants into high, medium, and low priority levels. In the high and medium groups, the variants are given a priority value and a reason for grading. All variants are initially in the low group. When a variant meets the preset pathogenicity criteria, it can be classified as a higher-level variant; the REVEL tool is used to predict the functional impact of SNP variants, and the SpliceAI tool predicts the splicing activity impact of variants; CNV TM The exon tool performs DNA copy number variation (CNV) analysis (resolution of ≥1 exon).

[0107] (3) Interpretation of candidate gene variants

[0108] First, on the basis of ensuring the sequencing quality, the candidate variants of G6PD, EPB41, SPTA1, SPTB, PIEZO1, SLC4A1, HBA1, HBA2, HBZ, HBB, PKLR, CDAN1, CDIN1, KIF23, KLF1, and SEC23B genes are analyzed. If positive pathogenic / likely pathogenic variants (P / LP) are found, positive test results will be reported; if no pathogenic variants are found from the candidate variants, reportable variants will be found in all variants in the clinical exome range. The interpretation of sequence variants is based on the internationally authoritative "2015 ACMG Guidelines for Clinical Interpretation of Genetic Variations" (ACMG Guidelines for short) and various guidelines of the ClinGen Sequence Variant Interpretation (SVI) Working Group (SVI Guidelines for short); CNV variant interpretation is based on the "Joint Consensus on Technical Standards for Reporting Interpretation of Primary Copy Number Variations" (PMID: 31690835) issued by ACMG and ClinGen in 2019. The various variants detected by the above sequencing are classified as pathogenic variants (P), likely pathogenic variants (LP), variants of uncertain significance (VUS), likely benign variants (LB), and benign variants (B). Clinical reports include pathogenic variants (P), likely pathogenic variants (LP), and variants of uncertain significance (VUS) that can explain the phenotype and family history of the subject.

[0109] Example 2: New pathogenic variant of G6PD gene NM_001042351.3: c.521G>C (p.Gly174Ala) found in a case of glucose-6-phosphate dehydrogenase deficiency

[0110] Proband 1, male, 1 month old, was found to have yellow skin 2 days after birth. The clinical diagnosis was hyperbilirubinemia, neonatal infection, G6PD deficiency, and neonatal kernicterus. The ratio method was used to detect the G6PD / 6PGD ratio = 0.07, indicating that the activity of glucose-6-phosphate dehydrogenase was significantly reduced. The pedigree is shown in the figure below. Figure 1 shown.

[0111] In this example, the customized clinical exome capture kit (TWIST) high-throughput sequencing was used to analyze the genomic variation information of the samples. Among the candidate genes for glucose-6-phosphate dehydrogenase deficiency, G6PD, EPB41, SPTA1, SPTB, PIEZO1, SLC4A1, HBA1, HBA2, HBZ, HBB, PKLR, CDAN1, CDIN1, KIF23, KLF1, and SEC23B, it was first found that proband 1 carried 3 sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( Figure 2). The variants are: G6PD (NM_001042351.3): c.521G>C; PIEZO1 (NM_001142864.4): c.5636A>G; PIEZO1 (NM_001142864.4): c.3076C>G. The pathogenicity rating of the above variants was performed according to the ACMG guidelines (see the table below). Among them, the PIEZO1(NM_001142864.4):c.5636A>G heterozygous variant was classified as a benign variant (B) and was excluded first; the PIEZO1(NM_001142864.4):c.3076C>G heterozygous variant was classified as a variant of unknown significance (VUS). After subsequent analysis, the blood examination of proband 1 did not find the typical stomatocytic manifestation of PIEZO1, so it was weakly correlated with the patient's phenotype and was excluded; the G6PD(NM_001042351.3):c.521G>C / p.Gly174Ala hemizygous variant was classified as a pathogenic variant (P) and was a candidate pathogenic variant of the patient.

[0112]

[0113] Specifically, the hemizygous variant c.521G>C (p.Gly174Ala) of the G6PD (NM_001042351.3) gene has the following original BAM file: Figure 3 As shown, the 521st base in the coding region mutated from G to C, and the 174th amino acid in the encoded protein mutated from glycine to alanine. According to the ACMG guidelines, this variant is classified as a pathogenic variant. The relevant evidence for this variant is PM1+PM2+PP3_Strong+PP4_Moderate, specifically:

[0114] PM1: This variant is located in the hotspot variant region. The HGMD database contains multiple amino acid variants in this region, all of which are DM (p.Pro172Ser, p.Phe173Leu, p.Asp176Gly, p.Ser179Cys / Asn, p.Asp181Val) (PubMed: 10556177, 9410474, 8193373, 22770933).

[0115] PM2: gnomAD v[2.1.1] shows that this variant is not currently included.

[0116] PP3_Strong: REVEL value is 0.978 (>0.932).

[0117] PP4_Moderate: The subject's phenotype is consistent with glucose-6-phosphate dehydrogenase deficiency, with glucose-6-phosphate dehydrogenase activity less than 10%.

[0118] The variant (NM_001042351.3:c.521G>C) has not been included in HGMD and is the first pathogenic variant discovered.

[0119] Therefore, the G6PD gene mutant NM_001042351.3:c.521G>C (p.Gly174Ala) was discovered by high-throughput sequencing technology after excluding other G6PD pathogenic genes. It is a new pathogenic variant associated with G6PD deficiency, and this variant can explain the G6PD deficiency phenotype of the proband, which is a diagnostic result for the proband. According to the population frequency, family information, computer bioinformatics analysis, clinical phenotype, etc., the results are accurate, expanding the G6PD gene pathogenic variant spectrum, and providing a basis for the diagnosis and genetic counseling of G6PD deficiency.

[0120] Example 3: New pathogenic variant of G6PD gene NM_001042351.3: c.661T>C (p.Phe221Leu) found in a case of glucose-6-phosphate dehydrogenase deficiency

[0121] Proband 2, male, 20 days old, clinically diagnosed with glucose-6-phosphate dehydrogenase deficiency, neonatal hyperbilirubinemia, neonatal hemolytic jaundice, neonatal hemolytic anemia, G2P2, 36+4 weeks, birth weight 2.76kg, admitted due to "skin yellowing for 15 days", jaundice recurred, blood routine: WBC: 9.2×10^9 / L, RBC: 2.36×10^12 / L, HGB: 84g / L, RCT: 3.74%; biochemical results: total bilirubin 401.2μmol / L, indirect bilirubin 388.5μmol / L, ALT: 12U / L, total bile acid: 15.23μmol / L, ratio method detection, G6PD / 6PGD ratio = 0.04, indicating that glucose-6-phosphate dehydrogenase activity is significantly reduced. Family chart as shown Figure 4 shown.

[0122] In this example, the genomic variation information of the samples was analyzed by high-throughput sequencing using a customized clinical exome capture kit (TWIST). Among the candidate genes for glucose-6-phosphate dehydrogenase deficiency, G6PD, EPB41, SPTA1, SPTB, PIEZO1, SLC4A1, HBA1, HBA2, HBZ, HBB, PKLR, CDAN1, CDIN1, KIF23, KLF1, and SEC23B, it was first found that proband 2 carried 2 sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( Figure 5). The sequence variations are: G6PD (NM_001042351.3): c.661T>C / p.Phe221Leu;

[0123] SPTA1(NM_003126.4):c.124G>A / p.Val42Ile. The pathogenicity rating of the above variants was performed according to the ACMG guidelines (see the table below). Among them, the heterozygous variant of SPTA1(NM_003126.4):c.124G>A / p.Val42Ile was classified as a variant of unknown significance (VUS). After subsequent analysis, the blood examination of proband 1 did not find the typical oval or other heterosexual red blood cell manifestations in SPTA1 disease, so it was weakly correlated with the patient's phenotype and was excluded.

[0124] The G6PD(NM_001042351.3):c.661T>C / p.Phe221Leu variant was classified as a possible pathogenic variant (LP) and was a candidate pathogenic variant in the patient.

[0125]

[0126] Specifically, the hemizygous variant c.661T>C / p.Phe221Leu of the G6PD (NM_001042351.3) gene, the original BAM file of its NGS is as follows Figure 6 As shown, the 661st base in the coding region mutated from T to C, and the mutation caused the 221st amino acid of the encoded protein to mutate from phenylalanine to leucine. According to the ACMG guidelines, this variant is classified as a possible pathogenic variant. The relevant evidence for this variant is PM1+PM2+PP3_Moderate+PP4_Moderate, specifically:

[0127] PM1: This variant is located in the hotspot variant region. The HGMD database contains multiple amino acid variants in this region, all of which are DM (p.Phe216Leu, p.Arg219Gly, p.Ile220Met, p.Ile224Phe, p.Trp225Cys) (PubMed: 2222408, 38066190, 21637675, 34533603, 31278024).

[0128] PM2: gnomAD v[2.1.1] shows that this variant is not currently included.

[0129] PP3_Moderate: REVEL value is 0.806 (>0.773).

[0130] PP4_Moderate: The subject's phenotype is consistent with glucose-6-phosphate dehydrogenase deficiency, and the glucose-6-phosphate dehydrogenase activity is less than 10%.

[0131] The variant (NM_001042351.3:c.661T>C) has not been included in HGMD and is the first pathogenic variant discovered.

[0132] Therefore, the G6PD gene mutant NM_001042351.3:c.661T>C (p.Phe221Leu) was discovered by high-throughput sequencing technology after excluding other G6PD pathogenic genes. It is a new pathogenic variant associated with G6PD deficiency, and this variant can explain the G6PD deficiency phenotype of the proband. It is a diagnostic result for the proband. According to the G6PD deficiency diagnosis and treatment guidelines, the G6PD gene pathogenic variant is the gold standard for the diagnosis of G6PD deficiency. The results are accurate based on population frequency, family information, computer bioinformatics analysis, clinical phenotype, etc., which expands the G6PD gene pathogenic variant spectrum and provides a basis for the diagnosis and genetic counseling of G6PD deficiency.

[0133] Example 4: New pathogenic variant of G6PD gene NM_001042351.3: c.680G>C (p.Arg227Pro) found in a case of glucose-6-phosphate dehydrogenase deficiency

[0134] Proband 3, male, 27 days old, G2P2, clinically diagnosed with glucose-6-phosphate dehydrogenase deficiency, clinical manifestations of hemolytic anemia, skin yellowing with anemia for 6 days, skin yellowing appeared 1 hour after birth, Hb lowest 73.8g / L, amniotic fluid third degree contamination, reticulocyte ratio 26.8%, newborn hemolytic disease test negative, does not support blood type incompatibility hemolytic disease, red blood cell fragility test indicates that the initial hemolysis concentration is 0.52, the liver and spleen are not large, the ratio method test, the G6PD / 6PGD ratio is close to 0, indicating that the activity of glucose-6-phosphate dehydrogenase is lost. The pedigree is shown in the figure below. Figure 7 shown.

[0135] In this example, the customized clinical exome capture kit (TWIST) high-throughput sequencing was used to analyze the genomic variation information of the samples. Among the candidate genes for glucose-6-phosphate dehydrogenase deficiency, G6PD, EPB41, SPTA1, SPTB, PIEZO1, SLC4A1, HBA1, HBA2, HBZ, HBB, PKLR, CDAN1, CDIN1, KIF23, KLF1, and SEC23B, it was first found that the proband carried 3 sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( Figure 8 ). The sequence variations are: G6PD (NM_001042351.3): c.680G>C / p.Arg227Pro;

[0136] EPB41 (NM_004437.4): c.1282-9C>T / p.?; PIEZO1 (NM_001142864.4): c.5215-24C>T / p.?. The pathogenicity rating of the above variants was performed according to the ACMG guidelines (see the table below). Among them,

[0137] EPB41(NM_004437.4):c.1282-9C>T / p.? The variant was classified as a benign variant (B) and was excluded first;

[0138] The heterozygous variant of PIEZO1 (NM_001142864.4): c.5215-24C>T / p.? was classified as a variant of unknown significance (VUS). After subsequent analysis, the blood examination of proband 1 did not reveal the typical stomatocytic manifestation of PIEZO1, so it was weakly correlated with the patient's phenotype and was excluded; G6PD (NM_001042351.3): c.680G>C / p.Arg227Pro variant was classified as a pathogenic variant (P) and was a candidate pathogenic variant of the patient.

[0139]

[0140] Specifically, the hemizygous variant c.680G>C (p.Arg227Pro) of the G6PD (NM_001042351.3) gene, the original BAM file of its NGS is as follows: Fig. 9 As shown, the 680th base in the coding region mutated from G to C, and the 227th amino acid in the encoded protein mutated from arginine to proline. According to the ACMG guidelines, this variant is classified as a pathogenic variant. The relevant evidence for this variant is: PM1+PM5+PM2+PP3_Moderate+PP4_Moderate.

[0141] PM1: This variant is located in the hotspot variant region. The HGMD database contains multiple amino acid variants in this region, all of which are DM (p.Trp225Cys, p.Arg227Leu / Gln / Asn, p.Asp228Asn, p.Ala231Pro, p.Cys232Tyr / Leu) (PubMed: 31278024, 2572288, 9192788, 31489982).

[0142] PM2: gnomAD v[2.1.1] shows that this variant is not currently included.

[0143] PM5: Other amino acid changes at the same amino acid site of this variant (p.Arg227Trp, p.Arg227Leu, p.Arg227Gln) have been reported in patients with glucose-6-phosphate dehydrogenase deficiency (PubMed: 10571945, 2572288, 1611091).

[0144] PP3_Moderate: REVEL value is 0.908 (>0.773).

[0145] PP4_Moderate: The subject's phenotype is consistent with glucose-6-phosphate dehydrogenase deficiency, and the glucose-6-phosphate dehydrogenase activity is close to 0.

[0146] The variant (NM_001042351.3:c.661T>C) has not been included in HGMD and is the first pathogenic variant discovered.

[0147] Therefore, the G6PD gene mutant c.680G>C (p.Arg227Pro) was discovered by high-throughput sequencing technology after excluding other G6PD pathogenic genes. It is a new pathogenic variant associated with G6PD deficiency, and this variant can explain the G6PD deficiency phenotype of the proband, which is a diagnostic result for the proband. According to the population frequency, computer bioinformatics analysis, clinical phenotype, etc., the results are accurate, expanding the G6PD gene pathogenic variant spectrum, and providing a basis for the diagnosis and genetic counseling of G6PD deficiency.

[0148] Example 6 Primer and probe composition and kit for detecting G6PD gene mutation sites in glucose-6-phosphate dehydrogenase deficiency

[0149] (1) The primer and probe combination and kit for detecting the G6PD gene mutation site of glucose-6-phosphate dehydrogenase deficiency are used to screen or diagnose glucose-6-phosphate dehydrogenase deficiency; they include specific primers and TaqMan probe combinations for the G6PD gene c.521G>C, c.661T>C, and c.680G>C mutation sites, respectively.

[0150] The specific primer and probe combination was designed using Primer 6.0 software, specifically:

[0151] 1) Targeting the G6PD gene c.521G>C site:

[0152] Forward primer A: 5′-CTCTGATCCTCACTCCCCGAA-3′;

[0153] Reverse Primer A: 5'-TCACCCTTGTCTGAGTTCTGGA-3'

[0154] G6PD gene wild-type probe Aw: 5′-AGGTCCCTCCCGAAGGGCTT-3′;

[0155] G6PD gene mutation probe Am: 5'-AGGTCCCTCGCGAAGGGCTT-3'.

[0156] 2) Targeting the G6PD gene c.661T>C site:

[0157] Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′;

[0158] Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3'

[0159] G6PD gene wild-type probe Bw: 5′-ATGGGGCCGAAGATCCTGTTGG-3′;

[0160] G6PD gene mutation probe Bm: 5'-ATGGGGCCGAGGATCCTGTTGG-3'.

[0161] 3) Targeting the G6PD gene c.680G>C site:

[0162] Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′;

[0163] Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3'

[0164] G6PD gene wild-type probe Cw: 5′-GATGTTGTCCCGGTTCCAGATG-3′;

[0165] G6PD gene mutation probe Cm: 5'-GATGTTGTCCGGGTTCCAGATG-3'.

[0166] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0167] In the same reaction system, the 5' ends of the probes respectively carry different reporter fluorescent groups. For example, in reaction system A, the 5' ends of the six probes for detecting three sites are respectively modified with different identifiable fluorescent dyes, including but not limited to one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LCRed640, Snarf 1, AcidFuchsin, Cy5.5, LC Red670, LC Red705, etc.

[0168] (2) Construction of multiple reaction system scheme: In order to determine the feasibility of constructing a multiple reaction system with the above probes, the present invention evaluates the possible interaction between the probes through mutual interference experiments. A single site and two-site quantitative reaction system was constructed according to the following reaction system: 2× premix Taq TM Buffer (Tris-HCl, pH8.9; 20mM KCl; 100mMMgCl23mM, Bio-Rad Biotechnology (Beijing) Co., Ltd.) 15μL, Forward primer (10μmoL) 1μL, Reverse primer (10μmoL) 1μL, DNA 1μL, wild-type (w) TaqMan probe (10μmoL) 1μL, mutant (m) TaqMan probe (10μmoL) 1μL, add ddH2O to 30μL. Record the Ct value results of each reaction system. If the Ct value of the two-site reaction system is not statistically different from the Ct value of the single-site reaction system (p>0.05), the probes of the dual system do not affect each other; on the contrary, if there is a statistical difference (p≤0.05), the probes of the dual system have mutual interference. The specific results are shown in the following table: There is no mutual interference between probe A, probe B, and probe C. Determine that reaction system A includes probe A, probe B, and probe C.

[0169]

[0170] (3) The reagent components of the kit can be general PCR amplification reaction reagents, including buffer (ionic environment), dNTP, PCR polymerase, water or other PCR additives, including but not limited to the 2×premix Taq TM Buffer (LATaq TM Version 2.0, Bio-Rad Biotechnology (Beijing) Co., Ltd.) and RNase- and DNase-free water.

[0171] (4) The kit adopts a single-tube multiplex fluorescent PCR reaction system, which is as follows:

[0172] Reaction system A: G6PD gene c.521G>C site, c.661T>C site and c.680G>C site

[0173] <![CDATA[2×premixTaq TM Buffer]]> 15μL Forward primer (10 μmol) 2.μL (1μL each of Forward Primer A and B) Reverse primer (10 μmol) 2μL (1μL each of Reverse Primer A and B) TaqMan probe (10 μmol) 6μL (probes Aw, Bw, Cw; probes Am, Bm, Cm, 1μL each) Template DNA 1μL (≥10ng) RNase-free and DNase-free water 4μL Total volume 30μL

[0174] (4) The amplification reaction procedure is as follows:

[0175]

[0176] (5) Operational procedures of multiple probe reagents for detecting the c.521G>C, c.661T>C, and c.680G>C variant sites of the G6PD gene for glucose-6-phosphate dehydrogenase deficiency:

[0177] As described in Example 1, clinical tissue samples are collected from the proband or family members of glucose-6-phosphate dehydrogenase deficiency, including but not limited to EDTA / sodium citrate anticoagulated peripheral blood;

[0178] As described in Example 1, genomic DNA from clinical tissue samples was obtained and quality controlled;

[0179] Using genomic DNA samples, a multiplex fluorescent PCR reaction system was prepared, and an amplification reaction was performed on a fluorescent quantitative PCR instrument according to the above reaction procedure;

[0180] Result interpretation: After the fluorescence quantitative PCR is completed, the data analysis and processing are performed using the fluorescence quantitative PCR instrument supporting software to obtain the results of each subject. By judging whether there are amplification peaks of wild-type and mutant-type specific probes at each detection site of the subject ( Figure 10-12 ) and determine whether the subject carries the G6PD gene pathogenic site and genotype according to the standards in the table below.

[0181]

[0182]

[0183] Example 7 Performance study results of a kit for detecting G6PD gene mutation sites in glucose-6-phosphate dehydrogenase deficiency

[0184] (1) Analytical performance research plan

[0185] In order to study the analytical performance of the single-tube multiplex fluorescent TaqMan probe PCR detection kit for the G6PD gene mutation site of glucose-6-phosphate dehydrogenase deficiency in the present invention, 400 clinical samples were used as research objects for performance study in this example, including: 238 clinical samples of patients clinically diagnosed with glucose-6-phosphate dehydrogenase deficiency and subjected to NGS testing in Example 1, 6 clinical samples (EDTA anticoagulated peripheral blood) from the father and mother of probands 1-3, and 156 EDTA anticoagulated peripheral blood samples of normal control population or control population without clinical manifestations of hereditary hemolytic anemia sent to Furui Medical Testing Laboratory for NGS analysis.

[0186] The test results of all samples were compared with the NGS analysis results or Sanger sequencing verification results. The consistency, sensitivity and specificity of the kit of the present invention for detecting the c.521G>C, c.661T>C, and c.680G>C variant sites of the G6PD gene for glucose-6-phosphate dehydrogenase deficiency were calculated respectively.

[0187] The compliance rate is compared by testing clinical samples in parallel with the reference method. Reference methods include but are not limited to: gold standard methods, industry-recognized methods, and methods that have been verified to meet the requirements for clinical intended use (such as the same test methods used by ISO15189 accredited laboratories). This study selected the gold standard method (Sanger) and the industry-recognized method (high-depth NGS sequencing) for comparison with the single-tube multiplex fluorescent TaqMan probe PCR detection method of this study, recorded the test results, and drew the following table:

[0188]

[0189] The calculation formula is as follows:

[0190] Matching rate = (a+d) / (a+b+c+d)×100%

[0191] Sensitivity = a / (a+c)×100%

[0192] Specificity = d / (b+d)×100%

[0193] (2) The system and steps of Sanger sequencing verification:

[0194] The PCR amplification systems (20 μL) for the c.521G>C, c.661T>C, and c.680G>C variant sites were: 2×premixTaq TMBuffer 10μL, Forward primer (10μmoL) 1μL, Reverse primer (10μmoL) 1μL, ddH2O 6μL, DNA 2μL; the primers are the amplification primers corresponding to each variant site provided in Example 6 above; PCR reaction program: 95℃5min, 35 cycles (95℃5min, 95℃30s, 64℃45sec), 72℃10min, 4℃ storage. After PCR amplification, 1% agarose gel electrophoresis was used for detection. After gel cutting, the product was recovered with the "Ordinary Agarose Gel DNA Recovery Kit (DP209)", and the product was diluted to 10ng / μL, and the recovered product was purified with Taq enzyme. All PCR products were subjected to Sanger sequencing analysis on the ABI 3730XL (AppliedBiosystems) platform using amplification primers.

[0195] (3) Analyze performance research results

[0196] After evaluation of 400 clinical samples, the detection rate of the c.521G>C mutation site by the kit for detecting the G6PD gene mutation site of glucose-6-phosphate dehydrogenase deficiency of the present invention was 100% consistent with the gold standard or industry-recognized method, and the sensitivity and specificity of detecting the c.521G>C mutation were both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.13 ):

[0197]

[0198] After evaluation of 400 clinical samples, the detection rate of c.661T>C mutation site by the kit for detecting G6PD gene mutation site of glucose-6-phosphate dehydrogenase deficiency of the present invention was 100% consistent with the gold standard or industry-recognized method, and the sensitivity and specificity of detecting c.661T>C mutation were both 100%, as shown in the following table and Sanger sequencing verification results ( Fig.14 ):

[0199]

[0200] After evaluation of 400 clinical samples, the detection rate of c.680G>C mutation site by the kit for detecting G6PD gene mutation site of glucose-6-phosphate dehydrogenase deficiency of the present invention was 100% consistent with the gold standard or industry-recognized method, and the sensitivity and specificity of detecting c.680G>C mutation were both 100%, as shown in the following table and Sanger sequencing verification results ( Fig.15 ):

[0201]

[0202] The above describes an embodiment of the present embodiment, but the present embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present embodiment, ordinary technicians in this field can also make many forms, all of which are within the protection of the present embodiment.

Claims

1. A single-tube multiplex real-time fluorescence quantitative PCR detection kit for detecting G6PD gene mutation sites based on TaqMan probes, characterized in that: It includes a reaction system A; The reaction system A includes the detection primers and detection probes as shown below: Detection primers: Targeting the G6PD gene c.521G>C site: Forward primer A: 5′-CTCTGATCCTCACTCCCCGAA-3′; Reverse primer A: 5′-TCACCCTTGTCTGAGTTCTGGA-3′; Targeting the G6PD gene c.661T>C site: Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′; Reverse primer B: 5′-ATCAAGGAAGGACATGGTGATGC-3′; Targeting the G6PD gene c.680G>C site: Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′; Reverse primer B: 5′-ATCAAGGAAGGACATGGTGATGC-3′; Detection probe: Targeting the G6PD gene c.521G>C site: G6PD gene wild-type probe Aw: 5′-AGGTCCCTCCCGAAGGGCTT-3′; G6PD gene mutant probe Am: 5′-AGGTCCCTCGCGAAGGGCTT-3′; Targeting the G6PD gene c.661T>C site: G6PD gene wild-type probe Bw: 5′-ATGGGGCCGAAGATCCTGTTGG-3′; G6PD gene mutation probe Bm: 5′-ATGGGGCCGAGGATCCTGTTGG-3′; Targeting the G6PD gene c.680G>C site: G6PD gene wild-type probe Cw: 5′-GATGTTGTCCCGGTTCCAGATG-3′; G6PD gene mutant probe Cm: 5′-GATGTTGTCCGGGTTCCAGATG-3′; The 5' end of the probe carries a reporter fluorescent group, and the 3' end carries a quencher fluorescent group; in reaction system A, the 5' end of each probe carries a different reporter fluorescent group, and the reporter fluorescent groups may be different according to the spectral wavelength.

2. The detection kit according to claim 1, characterized in that: The reaction system A is 30 μL, and each 30 μL of reaction system A contains: 2×premix Taq TM Buffer 15μL, 10μmoL / L Forward primer A 1μL, 10μmoL / L Reverse primer A 1μL, 10μmoL / L Forward primer B 1μL, 10μmoL / L Reverse primer B 1μL, 10μmoL / L probe Aw 1μL, 10μmoL / L probe Am 1μL, 10μmoL / L probe Bw 1μL, 10μmoL / L probe Bm 1μL, 10μmoL / L probe Cw 1μL, 10μmoL / L probe Cm 1μL, template DNA 1μL (≥10ng), 4μL RNase and DNase-free water.

3. The detection kit according to claim 1, characterized in that: The reporter fluorescent group is one of FAM, SYBR, Fluorescein, SYPRO Orange, VIC, JOE, TET, HEX, TAMRA, Texas Red, Alexa Fluor 633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LCRed640, Snarf 1, AcidFuchsin, Cy5.5, LC Red670, and LC Red705, and the quenching fluorescent group is MGB.

4. The detection kit according to claim 1, characterized in that: When the TaqMan probe is used for single-tube multiplex real-time fluorescence quantitative PCR amplification, the amplification reaction procedure is as follows:

5. Use of a reagent for detecting a G6PD gene mutation site in the preparation of a glucose-6-phosphate dehydrogenase deficiency detection reagent, characterized in that: The G6PD gene variation site includes at least one of the following sites: G6PD gene c.521G>C site, G6PD gene c.661T>C site, G6PD gene c.680G>C site; The gene number of the wild-type G6PD gene in the NCBI database is: NM_001042351.3, Among them, the G6PD gene c.521G>C site is a hemizygous mutation, the 521st base in the G6PD gene coding region mutates from G to C, and the mutation causes the 174th amino acid of the encoded protein to mutate from glycine to alanine; The G6PD gene c.661T>C site is a hemizygous mutation, where the 661th base in the G6PD gene coding region mutates from T to C, causing the 221st amino acid in the encoded protein to mutate from phenylalanine to leucine; The c.680G>C site of the G6PD gene is a hemizygous mutation. The 680th base in the coding region of the G6PD gene mutates from G to C. The mutation causes the 227th amino acid of the encoded protein to mutate from arginine to proline.

6. The use according to claim 5, characterized in that: The reagents for detecting G6PD gene mutation sites are probes and primers for detecting G6PD gene mutation sites; The primers include primers for the G6PD gene c.521G>C site, the G6PD gene c.661T>C site, and the G6PD gene c.680G>C site, and the primers for the sites are as follows: Targeting the G6PD gene c.521G>C site: Forward primer A: 5′-CTCTGATCCTCACTCCCCGAA-3′; Reverse Primer A: 5'-TCACCCTTGTCTGAGTTCTGGA-3' Targeting the G6PD gene c.661T>C site: Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′; Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3' Targeting the G6PD gene c.680G>C site: Forward primer B: 5′-ACGAATTCCTCCAGAACTCAGAC-3′; Reverse primer B: 5'-ATCAAGGAAGGACATGGTGATGC-3'. The probes include probes for the G6PD gene c.521G>C site, the G6PD gene c.661T>C site, and the G6PD gene c.680G>C site. The probes for the sites are as follows: Targeting the G6PD gene c.521G>C site: G6PD gene wild-type probe Aw: 5′-AGGTCCCTCCCGAAGGGCTT-3′; G6PD gene mutation probe Am: 5'-AGGTCCCTCGCGAAGGGCTT-3', Targeting the G6PD gene c.661T>C site: G6PD gene wild-type probe Bw: 5′-ATGGGGCCGAAGATCCTGTTGG-3′; G6PD gene mutation probe Bm: 5'-ATGGGGCCGAGGATCCTGTTGG-3', Targeting the G6PD gene c.680G>C site: G6PD gene wild-type probe Cw: 5′-GATGTTGTCCCGGTTCCAGATG-3′; G6PD gene mutation probe Cm: 5'-GATGTTGTCCGGGTTCCAGATG-3'.

Images