Multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting neurofibroma I-type NF1 gene variation sites

Abstract

The invention relates to a multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, a probe and a kit for detecting neurofibroma I type NF1 gene variation sites, and provides four NF1 gene mutation sites found for the first time, the four variation sites are NF1 gene c.21002105delinsAG, c.4561dup, c.6421del and c.6797dup variation sites respectively, and the mutation sites of the NF1 gene c.21002105delinsAG, the c.4561dup, the c.6421del and the c.6797dup can be used for detecting neurofibroma I type NF1 gene mutation sites. Meanwhile, the invention also provides a specific primer, a probe composition and a kit for detecting the NF1 gene variation site of the neurofibromatosis I type for screening or diagnosing the neurofibromatosis I type. The NF1 gene pathogenic variation spectrum is expanded, and a basis is provided for diagnosis and genetic counseling of the neurofibroma type I. The kit disclosed by the invention can comprehensively cover four new pathogenic variation sites of the NF1 gene related to the neurofibroma type I, 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 neurofibromatosis type I NF1 gene mutation site. Background Art

[0002] Neurofibromatosis type 1 (NF1; MIM#162200) is a common autosomal dominant syndrome with an incidence of approximately 1 in 3000 (Evans et al., 2010; Kallionpaa et al., 2018; Miller et al., 2019; Uusitalo et al., 2015). The clinical manifestations of NF1 are diverse, including café-au-lait macules (CALMs), cutaneous and plexiform neurofibromas, axillary and inguinal freckles, optic pathway gliomas, iris Lisch nodules, and an increased risk of benign and malignant tumors. Although the National Institutes of Health (NIH) diagnostic criteria remain the basis for clinical diagnosis (1987 NIH Consensus Statement on Neurofibromatosis), NF1 is challenging to diagnose, especially in pediatric patients, due to its high clinical variability and age-dependent expression (DeBella et al., 2000; Ferner & Gutmann, 2013). In many cases, early manifestations may be limited to café-au-lait macules, while other clinical manifestations gradually emerge later in life. Therefore, molecular genetic testing is essential in confirming the diagnosis of NF1 in children who do not yet fully meet the NIH consensus criteria and in differentiating it from other diseases with similar phenotypes, such as rasopathy syndrome (Tidyman & Rauen, 2009).

[0003] Neurofibromatosis type I is caused by mutations in the NF1 gene located on chromosome 17q11.2, which encodes neurofibromin, an important regulator of the Ras signaling pathway. Due to the large size of the NF1 gene, the presence of homologous sequences, and the lack of mutation hotspots, mutation screening of the NF1 gene faces significant challenges (Zhang et al., 2015).

[0004] In-depth research on neurofibromatosis type I helps to understand its genetic basis and pathogenesis, and early intervention can reduce the risk of complications, especially malignant tumors, in patients with neurofibromatosis. Currently, according to the NIH diagnostic criteria for NF1, a positive test for NF1 gene mutation is the most important diagnostic basis for neurofibromatosis type I. Early diagnosis is not only crucial for genetic counseling for patients and their families, but also helps to develop targeted management and treatment plans and improve prognosis. With the development of genome sequencing technology, scientists continue to discover new mutants in the NF1 gene, many of which are considered to be associated with different neurofibromatosis type I phenotypes and symptom severity. Accurate detection and analysis of these new mutants can not only improve the accuracy of early diagnosis of neurofibromatosis type I, but also lay the foundation for the development of personalized treatment plans.

[0005] Therefore, the study of the mutation spectrum of the NF1 gene is crucial to understanding the etiology of neurofibromatosis type I. These studies have revealed the correlation and related mechanisms between gene mutations and phenotypes such as neurofibromatosis, growth and development, and skeletal deformities, providing clues for the development of potential therapeutic strategies. Although several known mutations have been identified in the NF1 gene, there are still many undiscovered mutation sites. Expanding the comprehensive understanding of NF1 gene mutants will provide solid support for future clinical diagnosis, treatment, and molecular mechanism research of neurofibromatosis type I. Summary of the invention

[0006] The purpose of the present invention is to provide four NF1 gene mutation sites discovered for the first time to enrich the variation spectrum of the NF1 gene. The four variation sites are c.2100_2105delinsAG, c.4561dup, c.6421del and c.6797dup variation sites of the NF1 gene. At the same time, the present invention also provides a specific primer and probe combination for detecting neurofibromatosis type I NF1 gene variation sites and a kit for screening or diagnosing neurofibromatosis type I; including specific primers and TaqMan probe combinations for the NF1 gene c.2100_2105delinsAG, c.4561dup, c.6421del and c.6797dup variation sites, respectively.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The specific primer and probe combination of the present invention is designed using Primer 5.0 software, specifically:

[0009] 1) Targeting the NF1 gene c.2100_2105delinsAG site:

[0010] Forward primer A: 5'-TGGTAGCATTATGTTGGTCCAGAT-3' (SEQ ID NO. 1);

[0011] Reverse primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3' (SEQ ID NO. 2);

[0012] NF1 gene wild-type probe Aw: 5'-CCCTGACACTGAAGCTGTTCTGGT-3' (SEQ ID NO. 3);

[0013] NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3' (SEQ ID NO. 4),

[0014] 2) Targeting the NF1 gene c.4561dup site:

[0015] Forward primer B: 5'-GCTGTTTACAAATCAGCTGACAG-3' (SEQ ID NO. 5);

[0016] Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3' (SEQ ID NO. 6);

[0017] NF1 gene wild-type probe Bw: 5'-GATGGCAACACTTCTTGCAT-3' (SEQ ID NO. 7);

[0018] NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3' (SEQ ID NO. 8);

[0019] 3) Targeting the NF1 gene c.6421del site:

[0020] Forward primer C: 5'-GACCATGTTCAGTTACCAGCAC-3' (SEQ ID NO. 9);

[0021] Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3' (SEQ ID NO. 10);

[0022] NF1 gene wild-type probe Cw: 5'-TACCCAAATTTTACTTGCTG-3' (SEQ ID NO. 11);

[0023] NF1 gene mutant probe Cm: 5'-TACCCAAATTTACTTGCTGT-3' (SEQ ID NO. 12);

[0024] 4) Targeting the NF1 gene c.6797dup site:

[0025] Forward primer D: 5'-GCTAGCTACCAAGATCACCATAGC-3' (SEQ ID NO. 13);

[0026] Reverse primer D: 5'-CACCCCAGAAAGTAAGCTCCATG-3' (SEQ ID NO. 14);

[0027] NF1 gene wild-type probe Dw: 5'-ACTTACAACAGTCAAGTTCT-3' (SEQ ID NO. 15); NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3' (SEQ ID NO. 16).

[0028] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0029] 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.

[0030] A detection kit for detecting neurofibromatosis type I NF1 gene mutation sites using a double-tube multiplex real-time fluorescence quantitative PCR technique based on TaqMan probes, comprising a reaction system A and a reaction system B;

[0031] The reaction system A includes the detection primers and detection probes as shown below:

[0032] Detection primers:

[0033] Targeting the NF1 gene c.2100_2105delinsAG site:

[0034] Forward primer A: 5′-TGGTAGCATTATGTTGGTCCAGAT-3′;

[0035] Reverse primer A: 5′-AGCTCCACCCAAAAGGAGTAG-3′;

[0036] Targeting the NF1 gene c.4561dup site:

[0037] Forward primer B: 5′-GCTGTTTACAAATCAGCTGACAG-3′;

[0038] Reverse primer B: 5′-ATCATATCTAACAAGTGGCCTGGT-3′;

[0039] Targeting the NF1 gene c.6797dup site:

[0040] Forward primer D: 5′-GCTAGCTACCAAGATCACCATAGC-3′;

[0041] Reverse primer D: 5′-CACCCCAGAAAGTAAGCTCCATG-3′;

[0042] Detection probe:

[0043] Targeting the NF1 gene c.2100_2105delinsAG site:

[0044] NF1 gene wild-type probe Aw: 5′-CCCTGACACTGAAGCTGTTCTGGT-3′;

[0045] NF1 gene mutant probe Am: 5′-CCCTGACACAGTGTTCTGGT-3′;

[0046] Targeting the NF1 gene c.4561dup site:

[0047] NF1 gene wild-type probe Bw: 5′-GATGGCAACACTTCTTGCAT-3′;

[0048] NF1 gene mutant probe Bm: 5′-GATGGCAACACCTTCTTGC-3′;

[0049] Targeting the NF1 gene c.6797dup site:

[0050] NF1 gene wild-type probe Dw: 5′-ACTTACAACAGTCAAGTTCT-3′;

[0051] NF1 gene mutant probe Dm: 5′-TTACAACAGGTCAAGTTCTG-3′;

[0052] 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 the different spectral wavelengths;

[0053] The reaction system A is 20 μL, and each 20 μL of reaction system A contains:

[0054] 2×premix Taq TM Buffer 10μL, 10μmoL / L Forward Primer A 0.5μL, 10μmoL / L Reverse Primer A 0.5μL, 10μmoL / L Forward Primer B 0.5μL, 10μmoL / L Reverse Primer B 0.5μL, 10μmoL / L Forward Primer D 0.5μL, 10μmoL / L Reverse Primer D 0.5μL, 10μmoL / L Probe Aw 0.5μL, 10μmoL / L Probe Am 0.5μL, 10μmoL / L Probe Bw 0.5μL, 10μmoL / L Probe Bm 0.5μL, 10μmoL / L Probe Dw 0.5μL, 10μmoL / L Probe Dm 0.5μL, template DNA 2μL (≥10ng), RNase- and DNase-free water 2μL.

[0055] The reaction system B includes the detection primers and detection probes as shown below:

[0056] Detection primers:

[0057] Targeting the NF1 gene c.6421del site:

[0058] Forward primer C: 5'-GACATGTTCAGTTACCAGCAC-3';

[0059] Reverse primer C: 5′-CCACTTGCACGTTGGAATATCT-3′;

[0060] Detection probe:

[0061] Targeting the NF1 gene c.6421del site:

[0062] NF1 gene wild-type probe Cw: 5′-TACCCAAATTTTACTTGCTG-3′;

[0063] NF1 gene mutant probe Cm: 5′-TACCCAAATTTACTTGCTGT-3′;

[0064] The 5' end of the probe carries a reporter fluorescent group, and the 3' end carries a quencher fluorescent group.

[0065] The reaction system B is 20 μL, and each 20 μL of reaction system B contains:

[0066] 2×premix Taq TM Buffer 10μL, 10μmoL / L Forward primer C 0.5μL, 10μmoL / L Reverse primer C 0.5μL, 10μmoL / L probe Cw 0.5μL, 10μmoL / L probe Cm 0.5μL, template DNA 2μL (≥10ng), RNase and DNase-free water 6μL.

[0067] 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 same reaction system (reaction system A, reaction system B), the 5' end of the probe carries different reporter fluorescent groups, and can be distinguished from each other by a fluorescent quantitative PCR instrument according to different spectral wavelengths.

[0068] In the detection kit, when the TaqMan probe is amplified in double tube multiplex real-time fluorescence quantitative PCR, the amplification reaction procedure is as follows:

[0069]

[0070] The present invention also provides the use of a reagent for detecting NF1 gene mutation sites in the preparation of a neurofibromatosis type I detection reagent, wherein the NF1 gene mutation site comprises at least one of the following sites: NF1 gene c.2100_2105delinsAG site, NF1 gene c.4561dup site, NF1 gene c.6421del site, NF1 gene c.6797dup site;

[0071] The gene number of the wild-type NF1 gene in the NCBI database is: NM_000267.3;

[0072] Among them, the c.2100_2105delinsAG site of the NF1 gene is a heterozygous mutation. The bases from 2100 to 2105 of the NF1 gene are deleted, and AG is inserted. The mutation causes a frameshift mutation in the amino acid encoding the protein from position 701 (glutamic acid mutates to valine). After 44 amino acids of the frameshift mutation, translation is terminated.

[0073] The c.4561dup site of the NF1 gene is a heterozygous mutation. The base at position 4561 of the NF1 gene is repeated. The mutation causes a frameshift mutation (leucine to proline) in the amino acid encoding the protein from position 1521. After 21 amino acids of the frameshift mutation, translation is terminated.

[0074] The c.6421del site of the NF1 gene is a heterozygous mutation, with a deletion of the 6421 base of the NF1 gene. The mutation causes a frameshift mutation (tyrosine to threonine) in the amino acid encoding the protein from position 2141. Translation terminates after 37 amino acids of the frameshift mutation.

[0075] The c.6797dup site of the NF1 gene is a heterozygous mutation. The base at position 6797 of the NF1 gene is repeated. The mutation causes a frameshift mutation (serine to arginine) in the amino acid encoding the protein from position 2266. After the frameshift mutation of 19 amino acids, translation is terminated.

[0076] The NF1 gene c.2100_2105delinsAG site, NF1 gene c.4561dup site, NF1 gene c.6421del site, and NF1 gene c.6797dup site, these NF1 gene variation sites were discovered by the present invention using high-throughput sequencing technology after excluding other neurofibromatosis pathogenic genes, and these variations have not yet been included in the gnomAD database, and are pathogenic variation sites discovered for the first time.

[0077] The reagent for detecting the NF1 gene mutation site is one or more of a probe and a primer for detecting the NF1 gene mutation site.

[0078] The primers include primers for at least one variant site among the NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site, and the primers for the sites are respectively as follows:

[0079] Targeting the NF1 gene c.2100_2105delinsAG site:

[0080] Forward primer A: 5′-TGGTAGCATTATGTTGGTCCAGAT-3′;

[0081] Reverse Primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3'

[0082] Targeting the NF1 gene c.4561dup site:

[0083] Forward primer B: 5′-GCTGTTTACAAATCAGCTGACAG-3′;

[0084] Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3'

[0085] Targeting the NF1 gene c.6421del site:

[0086] Forward primer C: 5'-GACATGTTCAGTTACCAGCAC-3';

[0087] Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3'

[0088] Targeting the NF1 gene c.6797dup site:

[0089] Forward primer D: 5′-GCTAGCTACCAAGATCACCATAGC-3′;

[0090] Reverse primer D: 5′-CACCCCAGAAAGTAAGCTCCATG-3′.

[0091] The probes include probes for at least one variant site among the NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site, and the probes for the sites are as follows:

[0092] Targeting the NF1 gene c.2100_2105delinsAG site:

[0093] NF1 gene wild-type probe Aw: 5′-CCCTGACACTGAAGCTGTTCTGGT-3′;

[0094] NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3',

[0095] Targeting the NF1 gene c.4561dup site:

[0096] NF1 gene wild-type probe Bw: 5′-GATGGCAACACTTCTTGCAT-3′;

[0097] NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3'

[0098] Targeting the NF1 gene c.6421del site:

[0099] NF1 gene wild-type probe Cw: 5′-TACCCAAATTTTACTTGCTG-3′;

[0100] NF1 gene mutant probe Cm: 5′-TACCCAAATTTACTTGCTGT-3′;

[0101] Targeting the NF1 gene c.6797dup site:

[0102] NF1 gene wild-type probe Dw: 5′-ACTTACAACAGTCAAGTTCT-3′;

[0103] NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3'.

[0104] Compared with the prior art, the advantages of the present invention are: the present invention uses high-throughput sequencing technology to discover for the first time four NF1 gene mutation sites on the basis of excluding other neurofibromatosis pathogenic genes: NF1 gene c.2100_2105delinsAG site, c.4561dup site, c.6421del site, c.6797dup site, these mutation sites are new pathogenic mutations associated with neurofibromatosis type I, according to population frequency, family information, computer bioinformatics analysis, inheritance pattern, clinical phenotype, etc., the results are accurate, the NF1 gene pathogenic mutation spectrum is expanded, and the diagnosis and genetic counseling of neurofibromatosis type I are provided. In addition, the present invention provides primers and probe compositions and kits for detecting neurofibromatosis type I NF1 gene mutation sites for screening or diagnosing neurofibromatosis type I; including specific primers and TaqMan probe compositions for NF1 gene c.2100_2105delinsAG, c.4561dup, c.6421del, c.6797dup mutation sites, respectively. Among them, the detection kit and detection method for detecting the NF1 gene mutation site of neurofibromatosis type I based on the double-tube multiplex real-time fluorescence quantitative PCR technology of the present invention can comprehensively cover the 4 new pathogenic mutation sites of the NF1 gene related to neurofibromatosis type I. The multiplex TaqMan real-time fluorescence quantitative PCR technology is short in time consumption and simple in operation. It only needs to add the DNA specimen of the subject to the two reaction systems (reaction system A and reaction system B) of the present invention respectively, and then detect it on the machine without gel electrophoresis, purification recovery and sequencing and other post-processing. The result is accurate and reliable, which can meet the current clinical urgent need for a simple, fast and accurate patient and prenatal sample diagnosis and detection method. In addition, the primers and probes designed by the present invention have strong specificity, can specifically detect the above-mentioned 4 pathogenic mutation sites discovered for the first time, realize the diagnosis of neurofibromatosis type I, and provide a basis for guiding clinical diagnosis and treatment and genetic counseling. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 It is the candidate sequence variation detected by high-throughput sequencing of proband 1 in Example 2.

[0106] Figure 2 This is a BAM file diagram of the high-throughput sequencing results of the c.2100_2105delinsAG variant of the NF1 gene of proband 1 in Example 2.

[0107] Figure 3 It is the candidate sequence variation detected by high-throughput sequencing of proband 2 in Example 3.

[0108] Figure 4 This is a BAM file diagram of the high-throughput sequencing results of the c.4561dup variant of the NF1 gene of proband 2 in Example 3.

[0109] Figure 5 It is the candidate sequence variation detected by high-throughput sequencing of proband 3 in Example 4.

[0110] Figure 6 This is a BAM file diagram of the high-throughput sequencing results of the c.6421del mutation of the NF1 gene of proband 3 in Example 4.

[0111] Figure 7 It is the candidate sequence variation detected by high-throughput sequencing of proband 4 in Example 5.

[0112] Figure 8 This is a BAM file diagram of the high-throughput sequencing results of the c.6797dup variant of the NF1 gene of proband 4 in Example 5.

[0113] Fig. 9 This is the analysis result of the Primerselect software in Example 6 on the interaction between probe C and probe A, probe B, and probe D.

[0114] Fig.10 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe of the NF1 gene c.2100_2105delinsAG site in Example 6; wherein, the right figure is the amplification curve result diagram of the NF1 gene c.2100_2105delinsAG site heterozygote; and the left figure is the amplification curve result diagram of the c.2100_2105 wild type.

[0115] Fig.11 It is the result diagram of real-time fluorescence quantitative PCR of TaqMan probe of NF1 gene c.4561dup site in Example 6, wherein the right figure is the result diagram of amplification curve of heterozygote of NF1 gene c.4561dup site; the left figure is the result diagram of amplification curve of c.4561 wild type.

[0116] Fig.12 It is the result diagram of real-time fluorescence quantitative PCR of TaqMan probe at c.6421del site of NF1 gene in Example 6, wherein the right figure is the result diagram of amplification curve of heterozygote at c.6421del site of NF1 gene; the left figure is the result diagram of amplification curve of c.6421 wild type.

[0117] Fig.13 It is the result diagram of real-time fluorescence quantitative PCR of TaqMan probe of NF1 gene c.6797dup site in Example 6, wherein the right figure is the result diagram of amplification curve of heterozygote of NF1 gene c.6797dup site; the left figure is the result diagram of amplification curve of wild type of c.6797 site.

[0118] Fig.14It is the Sanger sequencing result diagram of NF1:NM_000267.3:c.2100_2105delinsAG in Example 7; wherein the red box is the detection site.

[0119] Fig.15 It is the Sanger sequencing result diagram of NF1:NM_000267.3:c.4561dup in Example 7; wherein the red box is the detection site.

[0120] Fig.16 It is the Sanger sequencing result diagram of NF1:NM_000267.3:c.6421del in Example 7, wherein the red box is the detection site.

[0121] Fig.17 It is the Sanger sequencing result diagram of NF1:NM_000267.3:c.6797dup in Example 7, wherein the red box is the detection site. DETAILED DESCRIPTION

[0122] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0123] Example 1 Detection of pathogenic variants in patients with neurofibromatosis type I using the Customized Clinical Exome Capture Kit (TWIST)

[0124] This example uses 536 clinical peripheral blood samples with a clinical diagnosis of neurofibromatosis type I or suspected neurofibromatosis type I and sent to the Furui Medical Laboratory as the research subjects, and collects clinical symptoms and signs, laboratory tests, and imaging examinations of the patient's skin system, nervous system, growth and development history, intelligence, skeletal system, hearing, vision, etc. The proband of the candidate neurofibromatosis type I and some family members were subjected to a "customized clinical exome capture kit (TWIST)" high-throughput sequencing test, which includes the following steps in sequence:

[0125] (1) Sample collection and genomic DNA extraction.

[0126] 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 purity of the DNA was measured using Nanodrop one, and the OD260nm / OD280nm of the obtained genomic DNA was between 1.7 and 2.0. The concentration of the 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°C.

[0127] (2) High-throughput sequencing and bioinformatics analysis of neurofibromatosis type I gene mutations.

[0128] First, the extracted genomic DNA was fragmented using the KAPA HyperPlus Library Preparation Kit, 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 genes 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 sequencer (Illumina, San Diego, CA, USA) (sequencing depth ≥150X, 20X coverage ≥96%). In all sequenced samples, quality control analysis was performed before data interpretation to ensure that the 20X coverage of the main pathogenic genes NF1, NF2, and SPRED1 of neurofibromatosis reached 100%. If it did not reach 100%, the region that did not reach 20X needed to be supplemented by Sanger sequencing to ensure the detection rate of pathogenic variants. According to the statistics of Genereviews, an international authoritative database, the positive rate of NF1, NF2, and SPRED1 in all patients with isolated neurofibromatosis phenotype is 93.7%, and the positive rate in neurofibromatosis patients who meet the NIH diagnostic criteria is greater than 97%.

[0129] The secondary and tertiary analysis of sequencing data was completed using the ISoGenetic v1.2.6 system (Shanghai Fujun Gene Biotechnology Co., Ltd.). Specifically, the NGS sequencing results were aligned with the human reference genome UCSCNCBI37 / hg19 using Sentieon BWAv0.7.15 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; the sequence of the target region was analyzed using the Alamut-Batch standalone version v1.9. software to annotate variants (annotation databases include: dbSNP, gnomAD, 1000g, ClinVar, OMIM, HGMD Professional Edition, etc.); use filterAlamut.py to sort the annotated variants according to high, medium, and low priority. In the high and medium groups, give the variants a priority value and a grading reason. 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; use FATHMM, FATHMMMKL, METALR, METASVM, MUTATIONASSESSOR, MUTATIONTASTERAGVGD, AGVGD, LRT, PROVEAN, SIFT, REVEL, and SpliceAI software provided by the VarSome database to perform bioinformatics prediction of SNP function and splicing activity; CNV TM The exon tool performs DNA copy number variation (CNV) analysis (resolution of ≥1 exon).

[0130] (3) Interpretation of candidate gene variants

[0131] First, the candidate variants of NF1, NF2, and SPRED1 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 in the candidate variants, candidate reportable variants will be found in the candidate variants within 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 for short); the interpretation of CNV variants is based on the "2019 ACMG Interpretation and Reporting Technical Standards for Chromosome Copy Number Variations". 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). The clinical report includes pathogenic variants (P), likely pathogenic variants (LP), and variants of uncertain significance (VUS) that can explain the phenotype and family history of the subject.

[0132] Example 2: A new pathogenic variant of the NF1 gene was found in a case of neurofibromatosis type I: NM_000267.3: c.2100_2105delinsAG (p.Glu701Valfs*46)

[0133] Proband 1 is a male, 4 years and 5 months old, clinically diagnosed with neurofibromatosis, found to have growth retardation for about 2 years, weight 15.5kg. Generally speaking, the height is 100.0cm, the external genitalia is Tanner stage I, the testicles are 1-2 ml, and skin examination shows many cafe au lait spots. The child was born by cesarean section at full term, with a birth weight of about 3.55kg and a height of about 50cm. There is no delay in walking and talking, and he is prone to colds. He had a previous ventricular septal defect surgery, 74.8cm at 1 year old, 82cm at 2 years old, and 91cm at 3 years old. The father is 168cm, the mother is 158cm, and the mother and grandfather also have multiple cafe au lait spots.

[0134] In this example, the customized clinical exome capture kit (TWIST) high-throughput sequencing was used to analyze the genomic variation information of the samples. It was first found that the proband carried 2 heterozygous sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( Figure 1). The sequence variations are: NF1 gene heterozygous variation NM_000267.3:c.2100_2105delinsAG (p.Glu701Valfs*46) and NM_000267.3:c.*871del. First, according to the ACMG guidelines, the NF1 gene variation NM_000267.3:c.*871del is classified as a benign variation and is excluded first. The relevant classification basis is: The NF1 gene variation NM_000267.3:c.*871del is located in the UTR region. The relevant evidence of this variation is BS1 (frequency in African population is 4.1%, higher than the incidence rate) and BS2 (6 homozygous variations were found in African population). According to the ACMG guidelines, this variation is classified as a benign variation.

[0135] The NF1 gene heterozygous variant NM_000267.3:c.2100_2105delinsAG (p.Glu701Valfs*46) was named by merging the adjacent cis-arranged NM_000267.3:c.2105del and NM_000267.3:c.2100_2102del according to HGVS. The original BAM file of NGS is as follows Figure 2 As shown, the bases 2100 to 2105 of the coding region are deleted, and AG is inserted. The mutation causes the amino acid of the encoded protein to undergo a frameshift mutation from position 701 (glutamic acid mutates to valine). After 44 amino acids of the frameshift mutation, translation is terminated. According to the ACMG guidelines, this mutation is classified as a pathogenic mutation. The relevant evidence for this mutation is PVS1+PM2+PP1+PP4, specifically:

[0136] PVS1: This frameshift variant is located in exon 18 of the NF1 gene transcript NM_000267.3 (a total of 57 exons, encoding 2818 amino acids), resulting in out-of-frame transcription products and premature termination codons. This variant may cause protein truncation or activate nonsense-mediated mRNA degradation, resulting in loss of function of the protein product of this gene. Truncating variants downstream of this variant are known to be pathogenic variants (PubMed: 25525159, 30308447, 25403449, 9475595).

[0137] PP1: The variant was inherited from the affected mother and grandfather, confirming that the variant co-segregates with the disease and the family.

[0138] PM2: This variant is not currently included in the gnomAD database.

[0139] PP4: The patient's phenotype and family history meet the NIH diagnostic criteria for NF1.

[0140] Therefore, the NF1 gene mutant c.2100_2105delinsAG (p.Glu701Valfs*46) was discovered by high-throughput sequencing technology after excluding other neurofibromatosis pathogenic genes. It is a new pathogenic variant associated with neurofibromatosis type I, and this variant can explain the phenotype of neurofibromatosis type I inherited by the proband's family, 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 spectrum of NF1 gene pathogenic variants, and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0141] Example 3: A novel pathogenic variant of the NF1 gene NM_000267.3: c.4561dup (p.Leu1521Profs*22) was found in a case of neurofibromatosis type I

[0142] Proband 2 was a 1-year-old male who was clinically diagnosed with neurofibromatosis. He had been found to have coffee-colored spots all over his body for more than a year. After birth, scattered coffee-colored spots of varying sizes were found all over his body, which gradually increased (N>6). He denied a family history of neurofibromatosis.

[0143] In this example, the customized clinical exome capture kit (TWIST) high-throughput sequencing was used to analyze the genomic variation information of the samples. It was first found that the proband carried 2 heterozygous sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( Figure 3 The sequence variations are: NF1 gene heterozygous variation NM_000267.3:c.4561dup(p.Leu1521Profs*22) and NF2 gene heterozygous variation

[0144] NM_000268.3:c.*2780dup. First, according to the ACMG guidelines, the NF2 gene variant NM_000268.3:c.*2780dup is classified as a benign variant and is excluded first. The relevant classification basis is: the NF2 gene variant NM_000268.3:c.*2780dup is located in the UTR region, and the relevant evidence of this variant is BS1 (frequency 1.7% in the African population, higher than the incidence rate) and BS2 (2 homozygous variants were found in the African population). According to the ACMG guidelines, this variant is classified as a benign variant.

[0145] The original BAM file of NGS for the heterozygous variant of NF1 gene NM_000267.3:c.4561dup(p.Leu1521Profs*22) is as follows Figure 4As shown, the 4561st base of the coding region is repeated, and the mutation causes the amino acid of the encoded protein to undergo a frameshift mutation from the 1521st position (leucine mutates to proline). After 21 amino acids of the frameshift mutation, the translation is terminated. According to the ACMG guidelines, this mutation is classified as a pathogenic mutation. The relevant evidence for this mutation is PVS1+PM2+PS2, specifically:

[0146] PVS1: This frameshift variant is located in exon 34 of transcript NM_000267.3 (a total of 57 exons), resulting in a premature stop codon, which may activate nonsense-mediated mRNA degradation, thereby affecting the function of the protein product encoded by this gene. Multiple truncating variants of this gene have been reported as pathogenic variants (PubMed: 24789688, 28961165, 23656349, 22106164).

[0147] PS2: The gene was not detected in the peripheral blood of the parents of the subject, suggesting that it may be a new mutation.

[0148] PM2: This variant is not currently included in the gnomAD database.

[0149] Therefore, the NF1 gene mutant NM_000267.3:c.4561dup (p.Leu1521Profs*22) was discovered by high-throughput sequencing technology after excluding other neurofibromatosis-causing genes. It is a new pathogenic variant associated with neurofibromatosis type I, and this variant can explain the sporadic neurofibromatosis type I phenotype of the proband. It is a diagnostic result for the proband. According to the diagnosis and treatment guidelines for neurofibromatosis type I, the patient was finally diagnosed with neurofibromatosis type I from suspected neurofibromatosis type I. According to the population frequency, family information, computer bioinformatics analysis, clinical phenotype, etc., the results are accurate, expanding the spectrum of pathogenic variants of the NF1 gene, and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0150] Example 4: A new pathogenic variant of the NF1 gene NM_000267.3: c.6421del (p.Tyr2141Thrfs*38) was found in a case of neurofibromatosis type I

[0151] Proband 3 was a 2-month-old female who was clinically suspected of having neurofibromatosis. Brown spots were found all over her body for 1.5 months. They were present since birth and gradually increased in number (N>6). She denied any family history of neurofibromatosis.

[0152] In this example, the customized clinical exome capture kit (TWIST) high-throughput sequencing was used to analyze the genomic variation information of the samples. It was first found that the proband carried 3 heterozygous sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( Figure 5 ). The sequence variations are: NF1 gene heterozygous variation NM_000267.3:c.6421del(p.Tyr2141Thrfs*38), SPRED1 gene heterozygous variation NM_152594.2:c.*3976C>T, NF2 gene heterozygous variation NM_000268.3:c.*2780dup. First, according to the ACMG guidelines, NF2 gene variation NM_000268.3:c.*2780dup and SPRED1 gene heterozygous variation NM_152594.2:c.*3976C>T were excluded first. The relevant classification basis is as follows: the NF2 gene variant NM_000268.3:c.*2780dup is located in the UTR region. The relevant evidence for this variant is BS1 (frequency in African population is 1.7%, higher than the incidence rate) and BS2 (two homozygous variants were found in African population). According to the ACMG guidelines, this variant is classified as a benign variant. The SPRED1 gene heterozygous variant NM_152594.2:c.*3976C>T is located in the UTR region, with a priority of 9 (lowest level). The relevant evidence for this variant is BP4 (CADD and other bioinformatics tools predict it to be benign) + BP7 (synonymous or non-coding region variants are predicted by bioinformatics tools to not affect splicing), BS4 (inherited from the father who is not ill). According to the ACMG guidelines, this variant is classified as a possible benign variant.

[0153] The original BAM file of NGS for the heterozygous variant of NF1 gene NM_000267.3:c.6421del(p.Tyr2141Thrfs*38) is as follows Figure 6 As shown, the 6421st base of the coding region is deleted, and the mutation causes a frameshift mutation (tyrosine to threonine) in the amino acid of the encoded protein from the 2141th position. After the frameshift mutation of 37 amino acids, the translation is terminated. According to the ACMG guidelines, this variant is classified as a pathogenic variant. The relevant evidence of this variant is: PVS1+PS2+PM2.

[0154] PVS1: This frameshift variant is located in exon 42 (of 57 exons) of transcript NM_000267.3, resulting in a premature stop codon, which may activate nonsense-mediated mRNA degradation, thereby affecting the function of the protein product encoded by this gene. Multiple truncating variants of this gene have been reported as pathogenic variants (PubMed: 18643859, 29914388, 30014477, 30308447).

[0155] PS2: The gene was not detected in the peripheral blood of the parents of the subject, suggesting that it may be a new mutation.

[0156] PM2: This variant is not currently included in the gnomAD database.

[0157] Therefore, the NF1 gene mutant NM_000267.3:c.6421del (p.Tyr2141Thrfs*38) was discovered by high-throughput sequencing technology after excluding other neurofibromatosis-causing genes. It is a new pathogenic variant associated with neurofibromatosis type I, and this variant can explain the sporadic neurofibromatosis type I phenotype of the proband. It is a diagnostic result for the proband. According to the diagnosis and treatment guidelines for neurofibromatosis type I, the patient was finally diagnosed with neurofibromatosis type I from suspected neurofibromatosis type I. According to the population frequency, family information, computer bioinformatics analysis, clinical phenotype, etc., the results are accurate, expanding the spectrum of pathogenic variants of the NF1 gene, and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0158] Example 5: A new pathogenic variant of the NF1 gene NM_000267.3: c.6797dup (p.Ser2266Argfs*20) was found in a case of neurofibromatosis type I

[0159] Proband 4 was a 7-year-old female who was clinically diagnosed with neurofibromatosis. Café au lait spots on her skin were found 7 years ago. They were present since birth and gradually increased in number (N>6). MRI of the head showed high signal shadows (neurofibroma) in the cerebellum and basal ganglia. The patient denied a family history of neurofibromatosis.

[0160] In this example, the customized clinical exome capture kit (TWIST) high-throughput sequencing was used to analyze the genomic variation information of the samples. It was first found that the proband carried 1 heterozygous sequence variation (population frequency ≤ 5%) and 0 candidate CNV variation ( Figure 7 ). The sequence variation is a heterozygous variation of the NF1 gene: NM_000267.3:c.6797dup(p.Ser2266Argfs*20), and its original BAM file of NGS is as follows Figure 8 As shown, the 6797th base of the coding region is repeated, and the mutation causes a frameshift mutation (serine to arginine) in the amino acid of the encoded protein from the 2266th position. After the frameshift mutation of 19 amino acids, the translation is terminated. According to the ACMG guidelines, this variant is classified as a pathogenic variant. The relevant evidence of this variant is: PVS1+PS2+PM2+PP4.

[0161] PVS1: The frameshift variant is located in exon 45 (out of 57 exons) of the NF1 gene transcript NM_000267.3, resulting in a premature stop codon, which may activate nonsense-mediated mRNA degradation, thereby affecting the function of the gene-encoded protein product. Known truncating variants downstream of this variant have been reported as pathogenic variants (PubMed: 25525159, 30308447, 25403449, 9475595).

[0162] PS2: The gene was not detected in the peripheral blood of the parents of the subject, suggesting that it may be a new mutation.

[0163] PM2: This variant is not currently included in the gnomAD database.

[0164] PP4: The patient's phenotype and family history meet the NIH diagnostic criteria for NF1.

[0165] Therefore, the NF1 gene mutant NM_000267.3:c.6797dup (p.Ser2266Argfs*20) was discovered using high-throughput sequencing technology after excluding other neurofibromatosis-causing genes. It is a new pathogenic variant associated with neurofibromatosis type I, and this variant can explain the sporadic neurofibromatosis type I 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 spectrum of NF1 gene pathogenic variants, and providing a basis for the diagnosis and genetic counseling of neurofibromatosis type I.

[0166] Example 6 Primer and probe composition and kit for detecting mutation sites of neurofibromatosis type I NF1 gene

[0167] (1) The primer and probe combination and kit for detecting neurofibromatosis type I NF1 gene mutation sites are used for screening or diagnosing neurofibromatosis type I; they include specific primers and TaqMan probe combinations for the NF1 gene c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup mutation sites, respectively.

[0168] The specific primer and probe combination was designed using Primer 5.0 software, specifically:

[0169] 1) Targeting the NF1 gene c.2100_2105delinsAG site:

[0170] Forward primer A: 5′-TGGTAGCATTATGTTGGTCCAGAT-3′;

[0171] Reverse Primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3'

[0172] NF1 gene wild-type probe Aw: 5′-CCCTGACACTGAAGCTGTTCTGGT-3′;

[0173] NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3',

[0174] 2) Targeting the NF1 gene c.4561dup site:

[0175] Forward primer B: 5′-GCTGTTTACAAATCAGCTGACAG-3′;

[0176] Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3'

[0177] NF1 gene wild-type probe Bw: 5′-GATGGCAACACTTCTTGCAT-3′;

[0178] NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3'

[0179] 3) Targeting the NF1 gene c.6421del site:

[0180] Forward primer C: 5'-GACATGTTCAGTTACCAGCAC-3';

[0181] Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3'

[0182] NF1 gene wild-type probe Cw: 5′-TACCCAAATTTTACTTGCTG-3′;

[0183] NF1 gene mutant probe Cm: 5′-TACCCAAATTTACTTGCTGT-3′;

[0184] 4) Targeting the NF1 gene c.6797dup site:

[0185] Forward primer D: 5′-GCTAGCTACCAAGATCACCATAGC-3′;

[0186] Reverse primer D: 5'-CACCCCAGAAAGTAAGCTCCATG-3'

[0187] NF1 gene wild-type probe Dw: 5′-ACTTACAACAGTCAAGTTCT-3′;

[0188] NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3'

[0189] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0190] 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.

[0191] (2) Use bioinformatics software to analyze probe interactions and optimize combinations: Use the Primerselect software in DNAstar7.1 software to open the multiple probe files saved in the same file, then select the designed multiple probes two by two, and select "primer pair dimers" under the "report" menu to analyze the dimer structures "dimers" between the probes. The pop-up window will tell you the "dimers" formed between this pair of probes, and use the dG value to evaluate this pair of probe combinations (usually the worst dG value is given, and theoretically the larger the dG value, the better). For combinations that form "dimers", the prompt is "bad", that is, the result is "poor", otherwise it is "no effect", and dimer structures "dimers" may be formed. The analysis results are as follows: Fig. 9 As shown, there is potential mutual interference between the probes.

[0192] (3) Construction of multiple reaction system scheme: Since bioinformatics analysis suggests that there is a possibility of dimerization between the probes of the present invention, the multiple reaction system scheme was determined through mutual interference experiments. The quantitative reaction system of a single site and two sites was constructed according to the following reaction system: 2× premix Taq TM10μL of buffer, 0.5μl of Forward primer (10μmoL), 0.5μl of Reverse primer (10μmoL), 2μl of DNA, 0.5μl of wild-type (w) TaqMan probe (10μmoL), 0.5μl of wild-type (m) TaqMan probe (10μmoL), add ddH2O to make up to 20μL. Record the Ct value results of each reaction system. If there is no statistical difference between the Ct value of the two-site reaction system and the Ct value of the single-site reaction system (p>0.05), the probes of the binary system do not affect each other; conversely, if there is a statistical difference (p≤0.05), the probes of the binary system interfere with each other. The specific results are shown in the following table: Probe C interacts with probe A, probe B, and probe D, but probe A, probe B, and probe D do not interfere with each other. It is determined that reaction system A includes probe A, probe B, and probe D; reaction system B includes probe C.

[0193]

[0194] (4) The reagent components of the kit may be general PCR amplification reaction reagents, including buffer, ions, dNTPs, PCR polymerase, water or other PCR additives, including but not limited to the 2× premix Taq TM Buffer (LATaq TM Version 2.0) and RNase- and DNase-free water.

[0195] (5) The kit adopts a double-tube multiplex fluorescent PCR reaction system, which is as follows:

[0196] Reaction system A: NF1 gene c.2100_2105delinsAG site, c.4561dup site and c.6797dup site

[0197] <![CDATA[2×premixTaq TM Buffer]]> 10μL Forward primer (10 μmol) 1.5μL (0.5μL each of Forward Primer A, B, and D) Reverse primer (10 μmol) 1.5μL (0.5μL each for Reverse Primer A, B, and D) TaqMan probe (10 μmol) 3μL (probes Aw, Bw, Dw; probes Am, Bm, Dm, 0.5μL each) Template DNA 2μL (≥10ng) RNase-free and DNase-free water 2μL Total volume 20μL

[0198] Reaction system B: NF1 gene c.6421del site

[0199] <![CDATA[2×premixTaq TM Buffer]]> 10μL Forward primer (10 μmol) 0.5μL (Forward primer C 0.5μL) Reverse primer (10 μmol) 0.5μL (Reverse Primer C 0.5μL) TaqMan probe (10 μmol) 1μL (0.5μL each for probe Cw and Cm) Template DNA 2μL (≥10ng) RNase-free and DNase-free water 6μL Total volume 20μL

[0200] (4) The amplification reaction procedure is as follows:

[0201]

[0202] (5) Operational procedures of multiple probe reagents for detecting the c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup mutation sites of the NF1 gene for neurofibromatosis type I:

[0203] As described in Example 1, clinical tissue samples are collected from the proband or family members of neurofibromatosis, including but not limited to EDTA / sodium citrate anticoagulated peripheral blood;

[0204] As described in Example 1, genomic DNA from clinical tissue samples was obtained and quality controlled;

[0205] Using genomic DNA samples, a multiplex fluorescent PCR reaction system is configured, and an amplification reaction is performed on a fluorescent quantitative PCR instrument according to the above reaction procedure;

[0206] Result interpretation: After the fluorescence quantitative PCR is completed, the software is used to analyze the data and 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 10-13 ) and determine whether the subject carries the NF1 gene pathogenic site and genotype according to the standards in the table below.

[0207]

[0208] Example 7 Performance study results of a kit for detecting NF1 gene mutation sites in neurofibromatosis type I

[0209] (1) Analytical performance research plan

[0210] In order to study the analytical performance of the double-tube multiplex fluorescent TaqMan probe PCR detection kit for the variant sites of the neurofibromatosis type I NF1 gene in the present invention, 544 clinical samples were used as research objects for performance research in this example, including: 536 clinical samples for NGS detection in Example 1, and 8 clinical samples (EDTA anticoagulated peripheral blood) from family members of the proband 1-4. All positive samples were compared with the NGS analysis results and Sanger sequencing verification results. For samples that were not subjected to NGS analysis, Sanger sequencing verification was performed. The sensitivity (Sensitivity) and specificity (Specificity) of the kit of the present invention for detecting the variant sites of the neurofibromatosis type I NF1 gene c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup were calculated respectively, and the formula is:

[0211] Sensitivity = number of true positive results / (number of true positive results + number of false negative results)

[0212] Specificity = number of true negative results / (number of true negative results + number of false positive results)

[0213] (2) The system and steps of Sanger sequencing verification:

[0214] PCR amplification system (20 μl) for the c.2100_2105delinsAG, c.4561dup, c.6421del, and c.6797dup variant sites. Each PCR reaction was a single-site, single-tube amplification system. There were 4 amplification systems in total: 2× premix Taq TM Buffer 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, 65℃1min), 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.

[0215] (3) Analyze performance research results

[0216] The sensitivity and specificity of the kit for detecting the c.2100_2105delinsAG mutation site of the neurofibromatosis type I NF1 gene mutation site of the present invention are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.14 ):

[0217]

[0218] The sensitivity and specificity of the kit for detecting the c.4561dup mutation site of neurofibromatosis type I NF1 gene of the present invention are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.15 ):

[0219]

[0220] The sensitivity and specificity of the kit for detecting the c.6421del mutation site of neurofibromatosis type I NF1 gene of the present invention are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.16 ):

[0221]

[0222] The sensitivity and specificity of the kit for detecting the c.6797dup mutation site of neurofibromatosis type I NF1 gene of the present invention are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.17 ):

[0223]

[0224] 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 detection kit for detecting the mutation site of neurofibromatosis type I NF1 gene by double-tube multiplex real-time fluorescence quantitative PCR technology based on TaqMan probe, characterized in that: It includes reaction system A and reaction system B; The reaction system A includes the detection primers and detection probes as shown below: Detection primers: Targeting the NF1 gene c.2100_2105delinsAG site: Forward primer A: 5′-TGGTAGCATTATGTTGGTCCAGAT-3′; Reverse primer A: 5′-AGCTCCACCCAAAAGGAGTAG-3′; Targeting the NF1 gene c.4561dup site: Forward primer B: 5′-GCTGTTTACAAATCAGCTGACAG-3′; Reverse primer B: 5′-ATCATATCTAACAAGTGGCCTGGT-3′; Targeting the NF1 gene c.6797dup site: Forward primer D: 5′-GCTAGCTACCAAGATCACCATAGC-3′; Reverse primer D: 5′-CACCCCAGAAAGTAAGCTCCATG-3′; Detection probe: Targeting the NF1 gene c.2100_2105delinsAG site: NF1 gene wild-type probe Aw: 5′-CCCTGACACTGAAGCTGTTCTGGT-3′; NF1 gene mutant probe Am: 5′-CCCTGACACAGTGTTCTGGT-3′; Targeting the NF1 gene c.4561dup site: NF1 gene wild-type probe Bw: 5′-GATGGCAACACTTCTTGCAT-3′; NF1 gene mutant probe Bm: 5′-GATGGCAACACCTTCTTGC-3′; Targeting the NF1 gene c.6797dup site: NF1 gene wild-type probe Dw: 5′-ACTTACAACAGTCAAGTTCT-3′; NF1 gene mutant probe Dm: 5′-TTACAACAGGTCAAGTTCTG-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; The reaction system B includes the detection primers and detection probes as shown below: Detection primers: Targeting the NF1 gene c.6421del site: Forward primer C: 5'-GACATGTTCAGTTACCAGCAC-3'; Reverse primer C: 5′-CCACTTGCACGTTGGAATATCT-3′; Detection probe: Targeting the NF1 gene c.6421del site: NF1 gene wild-type probe Cw: 5′-TACCCAAATTTTACTTGCTG-3′; NF1 gene mutant probe Cm: 5′-TACCCAAATTTACTTGCTGT-3′; The 5' end of the probe carries a reporter fluorescent group, and the 3' end carries a quencher fluorescent group.

2. The detection kit according to claim 1, characterized in that: The reaction system A is 20 μL, and each 20 μL of reaction system A contains: 2×premix Taq TM Buffer 10μL, 10μmoL / L Forward Primer A 0.5μL, 10μmoL / L Reverse Primer A 0.5μL, 10μmoL / L Forward Primer B 0.5μL, 10μmoL / L Reverse Primer B 0.5μL, 10μmoL / L Forward Primer D 0.5μL, 10μmoL / L Reverse Primer D 0.5μL, 10μmoL / L Probe Aw 0.5μL, 10μmoL / L Probe Am 0.5μL, 10μmoL / L Probe Bw 0.5μL, 10μmoL / L Probe Bm 0.5μL, 10μmoL / L Probe Dw 0.5μL, 10μmoL / L Probe Dm 0.5μL, template DNA 2μL (≥10ng), RNase- and DNase-free water 2μL.

3. The detection kit according to claim 1, characterized in that: The reaction system B is 20 μL, and each 20 μL of reaction system B contains: 2×premix Taq TM Buffer 10μL, 10μmoL / L Forward primer C 0.5μL, 10μmoL / L Reverse primer C 0.5μL, 10μmoL / L probe Cw 0.5μL, 10μmoL / L probe Cm 0.5μL, template DNA 2μL (≥10ng), RNase and DNase-free water 6μL.

4. 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, Acid Fuchsin, Cy5.5, LC Red670, and LC Red705, and the quenching fluorescent group is MGB.

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

6. Use of a reagent for detecting NF1 gene mutation sites in the preparation of a reagent for detecting neurofibromatosis type I, characterized in that: NF1 gene mutation sites include the following: NF1 gene c.2100_2105delinsAG site, NF1 gene c.4561dup site, NF1 gene c.6421del site, NF1 gene c.6797dup site; The gene number of the wild-type NF1 gene in the NCBI database is: NM_000267.3, Among them, the c.2100_2105delinsAG site of the NF1 gene is a heterozygous mutation, where bases 2100 to 2105 of the NF1 gene are deleted and AG is inserted. The mutation causes a frameshift mutation (glutamic acid to valine) in the amino acid encoding the protein from position 701. After 44 amino acids of the frameshift mutation, translation stops. The c.4561dup site of the NF1 gene is a heterozygous mutation. The base at position 4561 of the NF1 gene is repeated. The mutation causes a frameshift mutation (leucine to proline) in the amino acid encoding the protein from position 1521. After 21 amino acids of the frameshift mutation, translation is terminated. The c.6421del site of the NF1 gene is a heterozygous mutation, with a deletion of the 6421 base of the NF1 gene. The mutation causes a frameshift mutation (tyrosine to threonine) in the amino acid encoding the protein from position 2141. Translation terminates after 37 amino acids of the frameshift mutation. The c.6797dup site of the NF1 gene is a heterozygous mutation. The base at position 6797 of the NF1 gene is repeated. The mutation causes a frameshift mutation (serine to arginine) in the amino acid encoding the protein from position 2266. After the frameshift mutation of 19 amino acids, translation is terminated.

7. The use according to claim 6, characterized in that: The reagents for detecting the NF1 gene mutation site are probes and primers for detecting the NF1 gene mutation site.

8. The use according to claim 7, characterized in that: The primers include primers for the NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site. The primers for the sites are as follows: Targeting the NF1 gene c.2100_2105delinsAG site: Forward primer A: 5′-TGGTAGCATTATGTTGGTCCAGAT-3′; Reverse Primer A: 5'-AGCTCCACCCAAAAGGAGTAG-3' Targeting the NF1 gene c.4561dup site: Forward primer B: 5′-GCTGTTTACAAATCAGCTGACAG-3′; Reverse primer B: 5'-ATCATATCTAACAAGTGGCCTGGT-3' Targeting the NF1 gene c.6421del site: Forward primer C: 5'-GACATGTTCAGTTACCAGCAC-3'; Reverse primer C: 5'-CCACTTGCACGTTGGAATATCT-3' Targeting the NF1 gene c.6797dup site: Forward primer D: 5′-GCTAGCTACCAAGATCACCATAGC-3′; Reverse primer D: 5′-CACCCCAGAAAGTAAGCTCCATG-3′.

9. The use according to claim 7, characterized in that: The probes include probes for the NF1 gene c.2100_2105delinsAG site, the NF1 gene c.4561dup site, the NF1 gene c.6421del site, and the NF1 gene c.6797dup site. The probes for the sites are as follows: Targeting the NF1 gene c.2100_2105delinsAG site: NF1 gene wild-type probe Aw: 5′-CCCTGACACTGAAGCTGTTCTGGT-3′; NF1 gene mutant probe Am: 5'-CCCTGACACAGTGTTCTGGT-3', Targeting the NF1 gene c.4561dup site: NF1 gene wild-type probe Bw: 5′-GATGGCAACACTTCTTGCAT-3′; NF1 gene mutant probe Bm: 5'-GATGGCAACACCTTCTTGC-3' Targeting the NF1 gene c.6421del site: NF1 gene wild-type probe Cw: 5′-TACCCAAATTTTACTTGCTG-3′; NF1 gene mutant probe Cm: 5′-TACCCAAATTTACTTGCTGT-3′; Targeting the NF1 gene c.6797dup site: NF1 gene wild-type probe Dw: 5′-ACTTACAACAGTCAAGTTCT-3′; NF1 gene mutant probe Dm: 5'-TTACAACAGGTCAAGTTCTG-3'.

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