Multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting Marfan syndrome FBN1 gene variation sites

Abstract

The invention relates to multiple fluorescent quantitative PCR (Polymerase Chain Reaction) primers, a probe and a kit for detecting FBN1 gene variation sites of Marfan syndrome. According to the invention, three kinds of FBN1 gene variation sites, namely an FBN1 gene c.4120del site, an FBN1 gene c.6820Tgt, an FBN1 gene c.4120del site and an FBN1 gene c.6820Tgt, are found for the first time; according to the FBN1 gene, the FBN1 gene c.70387039del site is introduced, the FBN1 gene pathogenic variation spectrum is expanded, and a basis is provided for diagnosis and genetic counseling of Marfan syndromes. The invention also provides a primer, a probe composition and a kit for detecting the Marfan syndrome FBN1 gene variation sites, and the primer, the probe composition and the kit are used for screening or diagnosing the Marfan syndrome, and the sequences of the primer and the probe are as shown in SEQ ID NO.1-12. The primer and the probe designed by the invention are high in specificity, can specifically detect the three pathogenic variation sites found for the first time, and can be used for screening or diagnosing the Marfan syndrome. The diagnosis of the Marfan syndrome is realized.

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 mutation site of the Marfan syndrome FBN1 gene. Background Art

[0002] Marfan syndrome (FBN1; MIM#134797) is a common autosomal dominant syndrome. Marfan syndrome is a systemic disorder with high clinical variability that affects muscle and other connective tissues, showing a wide phenotypic continuum, ranging from mild (manifesting Marfan syndrome features in only one or a few systems) to severe and rapidly progressive neonatal multi-organ disease. The main manifestations involve the muscles, eyes, bones, and cardiovascular systems. Muscle manifestations are muscle underdevelopment; ophthalmological manifestations include myopia (more than 50% of patients); lens dislocation (seen in about 60% of patients); and an increased risk of retinal detachment, glaucoma, and early cataracts. Skeletal manifestations include bone overgrowth and joint laxity; abnormally long limbs relative to the trunk (dolichostenomelia); rib overgrowth, which may cause the sternum to be concave (excavatum) or convex (ostrich chest); and mild to severe and progressive scoliosis. The major morbidity and early mortality in Marfan syndrome are cardiovascular related and include aortic root dilatation (dilatation at the sinuses of Valsalva, predisposing to aortic laceration and rupture), mitral valve prolapse (with or without regurgitation), tricuspid valve prolapse, and proximal pulmonary artery dilatation. Severe and prolonged mitral and / or aortic regurgitation may lead to left ventricular dysfunction and occasionally heart failure.

[0003] The diagnosis of Marfan syndrome can be made through family history and clinical phenotype scoring systems, such as typical manifestations such as aortic root dilatation (z score ≥ 2.0) and lens dislocation. However, due to its high clinical variability and age-dependent expression, the early manifestations of Marfan syndrome are often atypical, such as pectus carinatum and scoliosis. Therefore, molecular genetic testing is crucial in confirming FBN1 and distinguishing it from other diseases with similar phenotypes (such as Loeys-Dietz syndrome). Marfan syndrome is caused by mutations in the FBN1 gene located on chromosome 15q21.1. The FBN1 gene is large (>600kb) and contains 65 exons. Due to the lack of mutation hotspots, mutation screening of the FBN1 gene faces significant challenges.

[0004] In-depth research on Marfan syndrome helps to understand its genetic basis and pathogenesis. Early intervention can reduce the complications of Marfan syndrome patients, especially in the early stage of diagnosis or when progressive aortic dilatation occurs. Usually, drugs that reduce hemodynamic stress on the aortic wall are taken first, such as beta-blockers or combined use of angiotensin receptor blockers (ARBs). At present, positive FBN1 gene mutation detection is the most important diagnostic basis for Marfan syndrome. Since the type of FBN1 gene mutation is correlated with the severity of the disease and future progression, early diagnosis is not only crucial for genetic counseling for patients and their families, but also helps to formulate targeted management and treatment plans and improve the prognosis level. Accurate detection and analysis of these new mutants can not only improve the accuracy of early diagnosis of Marfan syndrome, but also lay the foundation for the development of personalized treatment plans and obtain more molecular genetic mechanism data on the correlation between mutations and phenotypes.

[0005] Therefore, the study of the variation spectrum of the FBN1 gene is crucial to understanding the etiology of Marfan syndrome. These studies have revealed the correlation and related mechanisms between gene mutations and phenotypes of the heart, eyes, skeletal system, etc. in patients with Marfan syndrome, providing clues for the development of potential treatment strategies. Although several known mutations have been identified in the FBN1 gene, there are still many undiscovered mutation sites. Expanding the comprehensive understanding of FBN1 gene mutants will provide solid support for future clinical diagnosis, treatment and molecular mechanism research of Marfan syndrome. Summary of the invention

[0006] The purpose of the present invention is to provide three FBN1 gene mutation sites discovered for the first time to enrich the variation spectrum of the FBN1 gene. The three variation sites are FBN1 gene c.4120del, c.6820T>G, and c.7038_7039del variation sites, respectively. The present invention also provides a primer and probe combination and a kit for detecting Marfan syndrome FBN1 gene variation sites for screening or diagnosing Marfan syndrome; including specific primers and TaqMan probe combinations for FBN1 gene c.4120del, c.6820T>G, and c.7038_7039del 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 6.0 software, specifically:

[0009] 1) Targeting the c.4120del site of the FBN1 gene:

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

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

[0012] FBN1 gene wild-type probe Aw: 5'-ATGCTGGCTGCACATATGGGT-3' (SEQ ID NO. 3),

[0013] FBN1 gene mutant probe Am: 5'-ATGCTGGCTGCCATATGGGTT-3' (SEQ ID NO. 4);

[0014] 2) Targeting the c.6820T>G site of the FBN1 gene:

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

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

[0017] FBN1 gene wild-type probe Bw: 5'-TCCACAGATGCACATATATGTG-3' (SEQ ID NO. 7), FBN1 gene mutant probe Bm: 5'-TCCACAGATGCCCATATATGTG-3' (SEQ ID NO. 8);

[0018] 3) Targeting the c.7038_7039del site of the FBN1 gene:

[0019] Forward primer C: 5'-CCTTTGAAGCCCTTGGTGATC-3' (SEQ ID NO. 9),

[0020] Reverse primer C: 5'-AAGTTGAAGACTCGCTGGAATG-3' (SEQ ID NO. 10);

[0021] FBN1 gene wild-type probe Cw: 5'-GATCTGACACATGTTTTGTAGC-3' (SEQ ID NO.11), FBN1 gene mutant probe Cm: 5'-CGATCTGACACATTTTGTAGCA-3' (SEQ ID NO.12).

[0022] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0023] 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.

[0024] A detection kit for detecting FBN1 gene mutation sites of Marfan syndrome based on TaqMan probe single-tube multiplex real-time fluorescence quantitative PCR technology, comprising a reaction system A;

[0025] The reaction system A includes the detection primers and detection probes as shown below:

[0026] Detection primers:

[0027] Targeting the FBN1 gene c.4120del site:

[0028] Forward primer A: 5'-CATGGGAAGTTTGAAGGCAAGTCA-3',

[0029] Reverse primer A: 5′-ACAGAGCTCTAGTGCCAAATGATG-3′;

[0030] Targeting the FBN1 gene c.6820T>G site:

[0031] Forward primer B: 5'-GATCATACTCAACAGAGCAGAAGG-3',

[0032] Reverse primer B: 5′-GGAATTCACAAATGATCACGAGGC-3′;

[0033] Targeting the FBN1 gene c.7038_7039del site:

[0034] Forward primer C: 5'-CCTTTGAAGCCCTTGGTGATC-3',

[0035] Reverse primer C: 5′-AAGTTGAAGACTCGCTGGAATG-3′;

[0036] Detection probe:

[0037] Targeting the FBN1 gene c.4120del site:

[0038] FBN1 gene wild-type probe Aw: 5'-ATGCTGGCTGCACATATGGGT-3',

[0039] FBN1 gene mutant probe Am: 5′-ATGCTGGCTGCCATATGGGTT-3′;

[0040] Targeting the FBN1 gene c.6820T>G site:

[0041] FBN1 gene wild-type probe Bw: 5'-TCCACAGATGCACATATATGTG-3',

[0042] FBN1 gene mutant probe Bm: 5′-TCCACAGATGCCCATATATGTG-3′;

[0043] Targeting the FBN1 gene c.7038_7039del site:

[0044] FBN1 gene wild-type probe Cw: 5'-GATCTGACACATGTTTTGTAGC-3',

[0045] FBN1 gene mutant probe Cm: 5′-CGATCTGACACATTTTGTAGCA-3′;

[0046] 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;

[0047] 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.

[0048] The reaction system A is 30 μL, and each 30 μL of reaction system A contains:

[0049] 2×premix Taq TM Buffer 15μL, 10μmoL / L Forward primer C 0.5μL, 10μmoL / L Reverse primer C 0.5μL, 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 probe Cw 1μL, 10μmoL / L probe Cm 1μL, 10μmoL / L probe Aw 1μL, 10μmoL / L probe Am 1μL, 10μmoL / L probe Bw 1μL, 10μmoL / L probe Bm 1μL, template DNA 1μL (≥10ng), 5μL RNase and DNase-free water.

[0050] 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:

[0051]

[0052] The present invention also provides the use of a reagent for detecting a FBN1 gene mutation site in preparing a Marfan syndrome detection reagent, wherein the FBN1 gene mutation site includes at least one of the following sites: a FBN1 gene c.4120del site, a FBN1 gene c.6820T>G site, and a FBN1 gene c.7038_7039del site;

[0053] The gene number of the wild-type FBN1 gene in the NCBI database is: NM_000138.5;

[0054] Among them, the c.4120del site of the FBN1 gene is a heterozygous mutation, and the 4120th base in the coding region of the FBN1 gene is deleted. The mutation causes a frameshift mutation in the amino acid of the encoded protein from position 1374 (cysteine ​​mutates to alanine). After 38 amino acids of the frameshift mutation, translation is terminated.

[0055] The c.6820T>G site of the FBN1 gene is a heterozygous mutation. The 6820th base in the coding region of the FBN1 gene mutates from T to G. The mutation causes the 2274th amino acid of the encoded protein to mutate from cysteine ​​to glycine.

[0056] The c.7038_7039del site of the FBN1 gene is a heterozygous mutation, with a deletion of bases 7038-7039 in the coding region of the FBN1 gene. The mutation causes a frameshift mutation in the amino acid of the encoded protein from position 2347 (methionine mutates to valine), and translation terminates after 18 amino acids of the frameshift mutation.

[0057] The FBN1 gene c.4120del site, the FBN1 gene c.6820T>G site, and the FBN1 gene c.7038_7039del site are several FBN1 gene variation sites that were discovered by the present invention using high-throughput sequencing technology after excluding other Marfan syndrome pathogenic genes. These variations have not yet been included in the gnomAD, HGMD, and ClinVar databases and are pathogenic variation sites discovered for the first time.

[0058] The invention discloses an application of a reagent for detecting a FBN1 gene mutation site in preparing a Marfan syndrome detection kit. The reagent for detecting a FBN1 gene mutation site is one or more of a probe and a primer for detecting a FBN1 gene mutation site.

[0059] The primers include primers for at least one of the variant sites of the FBN1 gene c.4120del site, the FBN1 gene c.6820T>G site, and the FBN1 gene c.7038_7039del site, and the primers for the sites are as follows:

[0060] Targeting the FBN1 gene c.4120del site:

[0061] Forward primer A: 5'-CATGGGAAGTTTGAAGGCAAGTCA-3',

[0062] Reverse primer A: 5'-ACAGAGCTCTAGTGCCAAATGATG-3;'

[0063] Targeting the FBN1 gene c.6820T>G site:

[0064] Forward primer B: 5'-GATCATACTCAACAGAGCAGAAGG-3',

[0065] Reverse primer B: 5'-GGAATTCACAAATGATCACGAGGC-3'

[0066] Targeting the FBN1 gene c.7038_7039del site:

[0067] Forward primer C: 5'-CCTTTGAAGCCCTTGGTGATC-3',

[0068] Reverse primer C: 5′-AAGTTGAAGACTCGCTGGAATG-3′.

[0069] The probes include probes for at least one variant site among the FBN1 gene c.4120del site, the FBN1 gene c.6820T>G site, and the FBN1 gene c.7038_7039del site, and the probes for the sites are as follows:

[0070] Targeting the FBN1 gene c.4120del site:

[0071] FBN1 gene wild-type probe Aw: 5'-ATGCTGGCTGCACATATGGGT-3',

[0072] FBN1 gene mutant probe Am: 5′-ATGCTGGCTGCCATATGGGTT-3′;

[0073] Targeting the FBN1 gene c.6820T>G site:

[0074] FBN1 gene wild-type probe Bw: 5'-TCCACAGATGCACATATATGTG-3',

[0075] FBN1 gene mutant probe Bm: 5′-TCCACAGATGCCCATATATGTG-3′;

[0076] Targeting the FBN1 gene c.7038_7039del site:

[0077] FBN1 gene wild-type probe Cw: 5'-GATCTGACACATGTTTTGTAGC-3',

[0078] FBN1 gene mutant probe Cm: 5'-CGATCTGACACATTTTGTAGCA-3'.

[0079] 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 three FBN1 gene mutation sites on the basis of excluding other Marfan syndrome pathogenic genes: FBN1 gene c.4120del site, FBN1 gene c.6820T>G site, FBN1 gene c.7038_7039del site, these mutation sites are new pathogenic mutations associated with Marfan syndrome, according to population frequency, family information, computer bioinformatics analysis, inheritance pattern, clinical phenotype, etc., the results are accurate, the FBN1 gene pathogenic mutation spectrum is expanded, and the diagnosis and genetic counseling of Marfan syndrome are provided. In addition, the present invention provides primers and probe compositions and kits for detecting Marfan syndrome FBN1 gene mutation sites for screening or diagnosing Marfan syndrome; including specific primers and TaqMan probe compositions for FBN1 gene c.4120del, c.6820T>G, c.7038_7039del mutation sites, respectively. Among them, the detection kit and detection method for detecting the FBN1 gene mutation site of Marfan syndrome based on the single-tube multiplex real-time fluorescence quantitative PCR technology of the present invention can comprehensively cover the three new pathogenic mutation sites of the FBN1 gene related to Marfan syndrome. The multiplex TaqMan real-time fluorescence quantitative PCR technology is short in time 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 detect it on the machine. There is no need for gel electrophoresis, purification recovery and 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 diagnosis and detection method. In addition, the primers and probes designed by the present invention have strong specificity and can specifically detect the above three pathogenic mutation sites discovered for the first time, realize the diagnosis of Marfan syndrome, and provide a basis for guiding clinical diagnosis and treatment and genetic counseling. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 It is the pedigree chart of proband 1 in Example 2, and the arrow indicates proband 1.

[0081] Figure 2 It is the candidate sequence variation detected by high-throughput sequencing of proband 1 in Example 2.

[0082] Figure 3 This is a BAM file diagram of the high-throughput sequencing results of the c.4120del mutation of the FBN1 gene of proband 1 in Example 2.

[0083] Figure 4 It is the pedigree chart of proband 2 in Example 3, and the arrow indicates proband 2.

[0084] Figure 5 It is the candidate sequence variation detected by high-throughput sequencing of proband 2 in Example 3.

[0085] Figure 6 This is a BAM file diagram of the high-throughput sequencing results of the c.6820T>G variant of the FBN1 gene of proband 2 in Example 3.

[0086] Figure 7 It is the pedigree chart of proband 3 in Example 4, and the arrow indicates proband 3.

[0087] Figure 8 It is the candidate sequence variation detected by high-throughput sequencing of proband 3 in Example 4.

[0088] Fig. 9 It is the result diagram of real-time fluorescence quantitative PCR of TaqMan probe at c.4120del site of FBN1 gene in Example 6, wherein the right figure is the result diagram of amplification curve of heterozygote at c.4120del site of FBN1 gene; the left figure is the result diagram of amplification curve of c.4120 wild type.

[0089] Fig.10 It is the result diagram of real-time fluorescence quantitative PCR of TaqMan probe of FBN1 gene c.6820T>G site in Example 6, wherein the right figure is the result diagram of amplification curve of heterozygote of FBN1 gene c.6820T>G site; the left figure is the result diagram of amplification curve of c.6820T wild type.

[0090] Fig.11 It is the result diagram of real-time fluorescence quantitative PCR of TaqMan probe at c.7038_7039del site of FBN1 gene in Example 6; wherein, the right figure is the result diagram of amplification curve of heterozygote at c.7038_7039del site of FBN1 gene; and the left figure is the result diagram of amplification curve of c.498T wild type.

[0091] Fig.12 It is the Sanger sequencing result diagram of FBN1:NM_000138.5:c.4120del in Example 7; wherein, the red arrow indicates the detection site c.4120del heterozygous variation, and the green arrow indicates the detection site wild type.

[0092] Fig.13 This is the Sanger sequencing result diagram of FBN1:NM_000138.5:c.6820T>G in Example 7, wherein the red box is the detection site.

[0093] Fig.14It is the Sanger sequencing result diagram of FBN1:NM_000138.5:c.7038_7039del in Example 7; wherein the red box is the detection site. DETAILED DESCRIPTION

[0094] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0095] Example 1 Detection of pathogenic variants in patients with Marfan syndrome using the Customized Clinical Exome Capture Kit (TWIST)

[0096] This example uses 93 clinical peripheral blood samples that were clinically diagnosed with Marfan syndrome and sent to the Furui Medical Laboratory as the research objects, and collects clinical symptoms and signs, laboratory tests, and imaging examinations of the patient's eyes, heart, growth and development history, and skeletal system. The "Customized Clinical Exome Capture Kit (TWIST)" high-throughput sequencing test was performed on the candidate Marfan syndrome proband and some family members, which includes the following steps in sequence:

[0097] (1) Sample collection and genomic DNA extraction.

[0098] 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.

[0099] (2) High-throughput sequencing and bioinformatics analysis of Marfan syndrome gene mutations.

[0100] First, the extracted genomic DNA was fragmented using a KAPA kit (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 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%). For all sequenced samples, quality control analysis of Marfan syndrome and the main pathogenic genes of the diseases that need to be differentially diagnosed in the international authoritative Genereviews database, including FBN1, ACTA2, FOXE3, LOX, MAT2A, MFAP5, MYH11, MYLK, PRKG1, TGFB3, BGN, CBS, COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, COL3A1, COL5A1, COL5A2, FBN2, and PLOD1, was performed before data interpretation to ensure that the 20X coverage of these genes reached 100%. If it did not reach 100%, the areas that did not reach 20X needed to be supplemented by Sanger sequencing to ensure the detection rate of pathogenic variants.

[0101] 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. 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).

[0102] (3) Interpretation of candidate gene variants

[0103] First, candidate variants of FBN1, ACTA2, FOXE3, LOX, MAT2A, MFAP5, MYH11, MYLK, PRKG1, TGFB3, BGN, CBS, COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, COL3A1, COL5A1, COL5A2, FBN2, and PLOD1 genes are analyzed. If positive pathogenic / likely pathogenic variants (P / LP) are found among them, a positive test result will be reported; if no pathogenic variants are found in the candidate variants, candidate reportable variants will be searched among the candidate variants in the clinical exome range. The interpretation of sequence variations is based on the internationally authoritative "2015 ACMG Guidelines for Clinical Interpretation of Genetic Variations" (ACMG Guidelines) and various guidelines of the ClinGen Sequence Variant Interpretation (SVI) Working Group (SVI Guidelines); the interpretation of CNV variations is based on the "Joint Consensus on Technical Standards for Reporting Interpretation of Primary Copy Number Variations" (PMID: 31690835) released by ACMG and ClinGen in 2019. The various variations 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.

[0104] (4) Genetic diagnosis results of patients with Marfan syndrome

[0105] Among 93 cases with a clinical diagnosis of Marfan syndrome, a total of 78 cases were reported to have positive diagnosis results by the "Customized Clinical Exome Capture Kit (TWIST)" high-throughput sequencing test, of which 72 cases were found to have pathogenic variants in the FBN1 gene, 3 cases were found to have pathogenic variants in the FBN2 gene, 2 cases were found to have pathogenic variants in the COL9A1 gene, and 1 case was found to have pathogenic variants in the COL11A1 gene, with a positive diagnosis rate of 83.9%.

[0106] Example 2: New pathogenic variant NM_000138.5:c.4120del (p.Cys1374Alafs*39) in FBN1 gene found in Marfan syndrome case

[0107] Proband 1, male, 24 years old, had a 20-year history of chest expansion and a 7-year history of spinal deformity. Physical examination showed: height 186cm, slender fingers, and myopia (550 degrees). Auxiliary examinations indicated the results of cardiac ultrasound in an external hospital: left ventricular enlargement, widened aortic sinus, mild regurgitation of the aortic valve, mitral valve and tricuspid valve. Family history showed high-risk characteristics: the patient's brother died suddenly at the age of 20, with slender fingers and pectus excavatum, height 175cm; the mother and aunt both had slender fingers and a height of 164cm; the aunt's two sons both had slender fingers and pectus excavatum, height 171-175cm. Taking into account the patient's manifestations and family history, the possibility of Marfan syndrome is highly suspected. The pedigree is shown in the figure below. Figure 1 shown.

[0108] In this example, the genomic variation information of the samples was analyzed by high-throughput sequencing using a customized clinical exome capture kit (TWIST). It was first found that the proband carried 3 heterozygous sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( FBN1 , ACTA2 , FOXE3 , LOX , MAT2A , MFAP5 , MYH11 , MYLK , PRKG1 , TGFB3 , BGN , CBS , COL2A1 , COL9A1 , COL9A2 , COL9A3 , COL11A1 , COL11A2 , COL3A1 , COL5A1 , COL5A2 , FBN2 , and PLOD1 . Figure 2 ). The sequences are: FBN1(NM_000138.5):c.4120del / p.Cys1374Alafs*39;

[0109] COL9A3 (NM_001853.3): c.1786+24G>A / p.? ; CBS (NM_000071.2): c.1552+734G>C / p.? 。 The pathogenicity rating of the above variants was performed according to the ACMF guidelines (see the table below). Among them,

[0110] CBS (NM_000071.2): c.1552+734G>C / p.? The variant was classified as a benign variant (B) and was excluded first; COL9A3 (NM_001853.3): c.1786+24G>A was classified as a variant of unknown significance (VUS). It is known that the COL11A1 gene is associated with the Marfan syndrome phenotype and the disease is recessive. The patient is a heterozygous carrier of VUS, so the correlation with the patient's phenotype is weak and was excluded; FBN1 (NM_000138.5): c.4120del / p.Cys1374Alafs*39 variant was classified as a pathogenic variant (P) and was a candidate pathogenic variant of the patient.

[0111]

[0112] The original BAM file of NGS of the heterozygous variant of FBN1 gene NM_000138.5:c.4120del / p.Cys1374Alafs*39 is as follows Figure 3 As shown, the 4120th base of the coding region is missing, and the mutation causes a frameshift mutation (cysteine ​​mutation to alanine) in the amino acid of the encoded protein from the 1374th position. After the frameshift mutation of 38 amino acids, 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+PP4, specifically:

[0113] PVS1: This frameshift variant is located in exon 34 of transcript NM_000138.5 (total 66 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. Heterozygous loss of function of the FBN1 gene is known to be a disease mechanism of Marfan syndrome (PubMed: 17657824, 19293843).

[0114] PM2: gnomAD v[2.1.1] shows that this variant is not currently included.

[0115] PP4: The patient's phenotype and family history meet the Ghent diagnostic criteria for Marfan syndrome.

[0116] This variant has not yet been included in the HGMD database and is the first pathogenic variant discovered.

[0117] Therefore, the FBN1 gene mutant NM_000138.5:c.4120del (p.Cys1374Alafs*39) was discovered by high-throughput sequencing technology after excluding other Marfan syndrome pathogenic genes. It is a new pathogenic variant associated with Marfan syndrome, and this variant can explain the Marfan syndrome phenotype of the proband and his family members, 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 FBN1 gene pathogenic variants, and providing a basis for the diagnosis and genetic counseling of Marfan syndrome.

[0118] Example 3: New pathogenic variant NM_000138.5: c.6820T>G (p.Cys2274Gly) in FBN1 gene found in Marfan syndrome case

[0119] Proband 2 is a 12-year-old male with a clinical diagnosis of Marfan syndrome. He was found to have a bulge in the anterior chest for 7 years with an annual growth rate of 9 cm. He had a history of lens displacement and slender limbs. Echocardiography showed widening of the aortic sinus, mild mitral regurgitation, and mild tricuspid regurgitation. Height 169.5cm (>P97), weight 52.2kg. His mother was 164cm tall with slender limbs. His grandfather died suddenly. The pedigree is shown in the figure below. Figure 4 shown.

[0120] In this example, the genomic variation information of the samples was analyzed by high-throughput sequencing using a customized clinical exome capture kit (TWIST). It was first found that the proband carried 3 heterozygous sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( FBN1 , ACTA2 , FOXE3 , LOX , MAT2A , MFAP5 , MYH11 , MYLK , PRKG1 , TGFB3 , BGN , CBS , COL2A1 , COL9A1 , COL9A2 , COL9A3 , COL11A1 , COL11A2 , COL3A1 , COL5A1 , COL5A2 , FBN2 , and PLOD1 . Figure 5 ). The sequences are: FBN1 (NM_000138.5): c.6820T>G / p.Cys2274Gly; FBN2 (NM_001999.4): c.7841-16G>T / p.?; FBN2 (NM_001999.4): c.297T>C / p.Pro99=. The pathogenicity rating of the above variants was performed according to the ACMF guidelines (see the table below). Among them, FBN2 (NM_001999.4): c.7841-16G>T / p.? and FBN2(NM_001999.4):c.297T>C / p.Pro99=variants were classified as benign variants (B) and were excluded first; FBN1(NM_000138.5):c.6820T>G / p.Cys2274Gly variants were classified as pathogenic variants (P) and were candidate pathogenic variants for the patient.

[0121]

[0122] The original BAM file of NGS of FBN1 gene NM_000138.5:c.6820T>G (p.Cys2274Gly) heterozygous variant is as follows Figure 6 As shown, the 6820th base in the coding region mutated from T to G, and the 2274th amino acid in the encoded protein mutated from cysteine ​​to glycine. According to the ACMG guidelines, this variant is classified as a pathogenic variant. The relevant evidence for this variant is: PM1+PM2+PM5+PP3_Strong+PP2+PP4.

[0123] PP3_Strong: REVEL value is 0.979 (>0.932, VCEP).

[0124] PM1: This variant is located in the highly conserved cbEGF-like domain of the FBN1 protein, and changes in the cysteine ​​at this position may affect protein folding (VCEP).

[0125] PM2: gnomAD v[2.1.1] shows that this variant is not currently included.

[0126] PM5: Other amino acid changes at the same amino acid position (p.Cys2274Ser, p.Cys2274Trp) have been reported as pathogenic variants associated with Marfan syndrome (PubMed: 23142374, 17657824).

[0127] PP2: It is clear that pathogenic missense variants of the FBN1 gene are a common pathogenic mechanism and the rate of benign missense variants is low.

[0128] PP4: The patient's phenotype and family history meet the Ghent diagnostic criteria for Marfan syndrome.

[0129] This variant has not yet been included in the HGMD database and is the first pathogenic variant discovered.

[0130] Therefore, the FBN1 gene mutant NM_000138.5:c.6820T>G (p.Cys2274Gly) was discovered by high-throughput sequencing technology after excluding other Marfan syndrome pathogenic genes. It is a new pathogenic variant associated with Marfan syndrome, and this variant can explain the Marfan syndrome phenotype of the proband and his family members. It is a diagnostic result for the proband. According to the Marfan syndrome diagnosis and treatment guidelines, the FBN1 gene pathogenic variant is the gold standard for the diagnosis of Marfan syndrome. According to the population frequency, family information, computer bioinformatics analysis, clinical phenotype, etc., the results are accurate, expanding the spectrum of FBN1 gene pathogenic variants, and providing a basis for the diagnosis and genetic counseling of Marfan syndrome.

[0131] Example 4: New pathogenic variant NM_000138.5:c.7038_7039del (p.Met2347Valfs*19) in FBN1 gene found in Marfan syndrome case

[0132] Proband 3 is a 12-year-old male with suspected Marfan syndrome. He was found to be tall and thin, with slender fingers, height: 170cm, right chest flat, positive thumb sign, positive wrist sign, and scoliosis. Cardiac ultrasound showed right coronary sinus, no coronary sinus hypertrophy, mild mitral and tricuspid valve regurgitation. Spine X-ray: scoliosis deformity. Family history denied. Family pedigree is shown in the figure below. Figure 7 shown.

[0133] In this example, the genomic variation information of the samples was analyzed by high-throughput sequencing using a customized clinical exome capture kit (TWIST). It was first found that the proband carried 6 heterozygous sequence variations (population frequency ≤ 5%) and 0 candidate CNV variations ( FBN1 , ACTA2 , FOXE3 , LOX , MAT2A , MFAP5 , MYH11 , MYLK , PRKG1 , TGFB3 , BGN , CBS , COL2A1 , COL9A1 , COL9A2 , COL9A3 , COL11A1 , COL11A2 , COL3A1 , COL5A1 , COL5A2 , FBN2 , and PLOD1 . Figure 8 ). The sequences are: FBN1(NM_000138.5):c.7038_7039del / p.Met2347Valfs*19;

[0134] COL11A2(NM_080680.3):c.2017-5T>G / p.? ;COL9A1(NM_001851.6):c.976-14T>C / p.? ;

[0135] FBN2 (NM_001999.4): c.6446-26T>C / p.? ;COL11A2 (NM_080680.3): c.4587G>A / p.Pro1529=;COL2A1 (NM_001844.5): c.*28C>T / p.? 。 The pathogenicity rating of the above variants was performed according to the ACMF guidelines (see the table below). Among them, COL11A2 (NM_080680.3): c.2017-5T>G / p.? ;FBN2 (NM_001999.4): c.6446-26T>C / p.? ;COL11A2(NM_080680.3):c.4587G>A / p.Pro1529=;COL2A1(NM_001844.5):c.*28C>T / p.? The variant was classified as benign variant (B) or likely benign variant (LB) and was excluded first;

[0136] COL9A1 (NM_001851.6): c.976-14T>C / p.? was classified as a variant of unknown significance (VUS). It is known that the COL9A1 gene-related diseases are weakly correlated with the patient's phenotype and were excluded; FBN1 (NM_000138.5): c.7038_7039del /

[0137] The p.Met2347Valfs*19 variant was classified as a pathogenic variant (P) and is a candidate pathogenic variant in the patient.

[0138]

[0139]

[0140] The FBN1 gene NM_000138.5:c.7038_7039del (p.Met2347Valfs*19) heterozygous mutation, that is, the 7038-7039 bases in the coding region are deleted. The mutation causes the amino acid of the encoded protein to undergo a frameshift mutation from position 2347 (methionine mutates to valine). After the frameshift mutation of 18 amino acids, the translation is terminated. According to the ACMG guidelines, this mutation is classified as a pathogenic mutation. The relevant evidence for this mutation is specifically: PVS1+PM2+PP4.

[0141] PVS1: This frameshift variant is located in exon 58 (out of 66) of transcript NM_000138.5, resulting in a premature stop codon that may activate nonsense-mediated mRNA degradation, thereby affecting the function of the protein product encoded by this gene. Heterozygous loss of function of the FBN1 gene is known to be a disease mechanism of Marfan syndrome (PubMed: 17657824, 19293843).

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

[0143] PP4: The patient's phenotype and family history meet the Ghent diagnostic criteria for Marfan syndrome.

[0144] This variant has not yet been included in the HGMD database and is the first pathogenic variant discovered.

[0145] Therefore, the FBN1 gene mutant c.7038_7039del (p.Met2347Valfs*19) was discovered by high-throughput sequencing technology after excluding other Marfan syndrome pathogenic genes. It is a new pathogenic variant associated with Marfan syndrome, and this variant can explain the sporadic Marfan syndrome 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 spectrum of FBN1 gene pathogenic variants, and providing a basis for the diagnosis and genetic counseling of Marfan syndrome.

[0146] Example 6 Primer and probe composition and kit for detecting FBN1 gene mutation sites in Marfan syndrome

[0147] (1) The primer and probe combination and kit for detecting the FBN1 gene mutation site of Marfan syndrome are used for screening or diagnosing Marfan syndrome; they include specific primers and TaqMan probe combinations for the FBN1 gene c.4120del, c.6820T>G, and c.7038_7039del mutation sites, respectively.

[0148] The specific primer and probe combination was designed using Primer 6.0 software, specifically:

[0149] 1) Targeting the c.4120del site of the FBN1 gene:

[0150] Forward primer A: 5'-CATGGGAAGTTTGAAGGCAAGTCA-3,

[0151] Reverse Primer A: 5'-ACAGAGCTCTAGTGCCAAATGATG-3'.

[0152] FBN1 gene wild-type probe Aw: 5'-ATGCTGGCTGCACATATGGGT-3',

[0153] FBN1 gene mutant probe Am: 5'-ATGCTGGCTGCCATATGGGTT-3'.

[0154] 2) Targeting the c.6820T>G site of the FBN1 gene:

[0155] Forward primer B: 5'-GATCATACTCAACAGAGCAGAAGG-3',

[0156] Reverse primer B: 5′-GGAATTCACAAATGATCACGAGGC-3′.

[0157] FBN1 gene wild-type probe Bw: 5'-TCCACAGATGCACATATATGTG-3',

[0158] FBN1 gene mutant probe Bm: 5'-TCCACAGATGCCCATATATGTG-3'.

[0159] 3) Targeting the c.7038_7039del site of the FBN1 gene:

[0160] Forward primer C: 5'-CCTTTGAAGCCCTTGGTGATC-3',

[0161] Reverse primer C: 5′-AAGTTGAAGACTCGCTGGAATG-3′.

[0162] FBN1 gene wild-type probe Cw: 5′-GATCTGACACATGTTTTGTAGC-3′;

[0163] FBN1 gene mutant probe Cm: 5'-CGATCTGACACATTTTGTAGCA-3'.

[0164] The 5' end of the probe carries a different reporter fluorescent group, and the 3' end carries an MGB quencher fluorescent group.

[0165] In the reaction system, the 5' end of the probe carries a different reporter fluorescent group. For example, in reaction system A, the 5' ends of the six probes for detecting three sites are 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 Fluor 633, ResoLight, EvaGreen, LC Green, Cy3, Cy5, Yellow555, LC Red610, ROX, SYPRO Ruby, LCRed640, Snarf 1, AcidFuchsin, Cy5.5, LC Red670, LC Red705, etc.

[0166] (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 15μL of buffer, 0.5μl of Forward primer (10μmoL), 0.5μl of Reverse primer (10μmoL), 1μl of DNA, 1μl of wild-type (w) TaqMan probe (10μmoL), 1μl of mutant (m) TaqMan probe (10μmoL), add ddH2O to make up to 30μL. 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; on the contrary, if there is a statistical difference (p≤0.05), there is interference between the probes of the binary system. The specific results are shown in the following table: There is no mutual interference between probes A, probe B, and probe C. Determine that reaction system A includes probes A, B, and C.

[0167]

[0168]

[0169] (3) 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.

[0170] (4) The kit adopts a single-tube multiplex fluorescent PCR reaction system, which is as follows:

[0171] Reaction system A: FBN1 gene c.4120del site, c.6820T>G site and c.7038_7039del site

[0172] <![CDATA[2×premixTaq TM Buffer]]> 15μL Forward primer (10 μmol) 1.5μL (0.5μL each of Forward Primer A, B, and C) Reverse primer (10 μmol) 1.5μL (0.5μL each for Reverse Primer A, B, and C) 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 5μL Total volume 30μL

[0173] (4) The amplification reaction procedure is as follows:

[0174]

[0175] (5) Operational procedures for multiple probe reagent detection of Marfan syndrome FBN1 gene c.4120del, c.6820T>G, c.7038_7039del mutation sites:

[0176] As described in Example 1, clinical tissue samples are collected from the proband or family members of Marfan syndrome, including but not limited to EDTA / sodium citrate anticoagulated peripheral blood;

[0177] As described in Example 1, genomic DNA from clinical tissue samples was obtained and quality controlled;

[0178] 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;

[0179] 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 9-11 ) and determine whether the subject carries the FBN1 gene pathogenic site and genotype according to the standards in the table below.

[0180]

[0181] Example 7 Performance study results of a kit for detecting FBN1 gene mutation sites in Marfan syndrome

[0182] (1) Analytical performance research plan

[0183] In order to study the analytical performance of the single-tube multiplex fluorescent TaqMan probe PCR detection kit for the Marfan syndrome FBN1 gene mutation site in the present invention, 140 clinical samples were used as research objects for performance research in this example, including: 93 clinical samples for NGS detection in Example 1, 8 clinical samples from family members of the proband 1-3 (EDTA anticoagulated peripheral blood), and the others were normal controls. All positive samples were compared with the NGS analysis results and Sanger sequencing verification results. Samples that were not subjected to NGS analysis were all subjected to Sanger sequencing verification. The sensitivity (Sensitivity) and specificity (Specificity) of the kit of the present invention for detecting the Marfan syndrome FBN1 gene c.4120del, c.6820T>G, and c.7038_7039del mutation sites were calculated respectively, and the formula was:

[0184] Sensitivity = number of true positive results / (number of true positive results + number of false negative results)

[0185] Specificity = number of true negative results / (number of true negative results + number of false positive results)

[0186] (2) The system and steps of Sanger sequencing verification:

[0187] The PCR amplification systems (20 μl) for the c.4120del, c.6820T>G, and c.7038_7039del variant sites were: 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 product was purified and recovered with Taq enzyme. All PCR products were subjected to Sanger sequencing analysis on the ABI3730XL (Applied Biosystems) platform using amplification primers.

[0188] (3) Analyze performance research results

[0189] The sensitivity and specificity of the Marfan syndrome FBN1 gene mutation site detection kit of the present invention for detecting the c.4120del mutation site are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.12 ):

[0190]

[0191]

[0192] The sensitivity and specificity of the Marfan syndrome FBN1 gene mutation site detection kit of the present invention for detecting the c.6820T>G mutation site are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.13 ):

[0193]

[0194] The sensitivity and specificity of the Marfan syndrome FBN1 gene mutation site detection kit of the present invention for detecting the c.7038_7039del mutation site are both 100%, as shown in the following table and the Sanger sequencing verification results ( Fig.14 ):

[0195]

[0196] 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 Marfan syndrome FBN1 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 FBN1 gene c.4120del site: Forward primer A: 5'-CATGGGAAGTTTGAAGGCAAGTCA-3'; Reverse primer A: 5′-ACAGAGCTCTAGTGCCAAATGATG-3′; Targeting the FBN1 gene c.6820T>G site: Forward primer B: 5′-GATCATACTCAACAGAGCAGAAGG-3′; Reverse primer B: 5′-GGAATTCACAAATGATCACGAGGC-3′; Targeting the FBN1 gene c.7038_7039del site: Forward primer C: 5′-CCTTTGAAGCCCTTGGTGATC-3′; Reverse primer C: 5′-AAGTTGAAGACTCGCTGGAATG-3′; Detection probe: Targeting the FBN1 gene c.4120del site: FBN1 gene wild-type probe Aw: 5′-ATGCTGGCTGCACATATGGGT-3′; FBN1 gene mutant probe Am: 5′-ATGCTGGCTGCCATATGGGTT-3′; Targeting the FBN1 gene c.6820T>G site: FBN1 gene wild-type probe Bw: 5′-TCCACAGATGCACATATATGTG-3′; FBN1 gene mutant probe Bm: 5′-TCCACAGATGCCCATATATGTG-3′; Targeting the FBN1 gene c.7038_7039del site: FBN1 gene wild-type probe Cw: 5′-GATCTGACACATGTTTTGTAGC-3′; FBN1 gene mutant probe Cm: 5′-CGATCTGACACATTTTGTAGCA-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.

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 C 0.5μL, 10μmoL / L Reverse primer C 0.5μL, 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 probe Cw 1μL, 10μmoL / L probe Cm 1μL, 10μmoL / L probe Aw 1μL, 10μmoL / L probe Am 1μL, 10μmoL / L probe Bw1μL, 10μmoL / L probe Bm 1μL, template DNA 1μL (≥10ng), 5μL 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, Acid Fuchsin, 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 FBN1 gene mutation sites in the preparation of a Marfan syndrome detection reagent, characterized in that: The FBN1 gene mutation sites include the following sites: FBN1 gene c.4120del site, FBN1 gene c.6820T>G site, FBN1 gene c.7038_7039del site; The gene number of the wild-type FBN1 gene in the NCBI database is: NM_000138.5, Among them, the c.4120del site of the FBN1 gene is a heterozygous mutation, with a deletion of base 4120 in the coding region of the FBN1 gene. The mutation causes a frameshift mutation (cysteine ​​to alanine) in the amino acid of the encoded protein from position 1374. After 38 amino acids of the frameshift mutation, translation is terminated. The c.6820T>G site of the FBN1 gene is a heterozygous mutation. The 6820th base in the coding region of the FBN1 gene mutates from T to G. The mutation causes the 2274th amino acid of the encoded protein to mutate from cysteine ​​to glycine. The c.7038_7039del site of the FBN1 gene is a heterozygous mutation, with a deletion of bases 7038-7039 in the coding region of the FBN1 gene. The mutation causes a frameshift mutation in the amino acid of the encoded protein from position 2347 (methionine mutates to valine), and translation terminates after 18 amino acids of the frameshift mutation.

6. Use of a reagent for detecting FBN1 gene mutation sites in the preparation of a Marfan syndrome detection kit, characterized in that: The reagents for detecting FBN1 gene mutation sites are probes and primers for detecting FBN1 gene mutation sites; The primers include primers for the FBN1 gene c.4120del site, the FBN1 gene c.6820T>G site, and the FBN1 gene c.7038_7039del site, and the primers for the sites are as follows: Targeting the FBN1 gene c.4120del site: Forward primer A: 5'-CATGGGAAGTTTGAAGGCAAGTCA-3'; Reverse Primer A: 5'-ACAGAGCTCTAGTGCCAAATGATG-3' Targeting the FBN1 gene c.6820T>G site: Forward primer B: 5′-GATCATACTCAACAGAGCAGAAGG-3′; Reverse primer B: 5'-GGAATTCACAAATGATCACGAGGC-3' Targeting the FBN1 gene c.7038_7039del site: Forward primer C: 5′-CCTTTGAAGCCCTTGGTGATC-3′; Reverse primer C: 5′-AAGTTGAAGACTCGCTGGAATG-3′; The probes include probes for the FBN1 gene c.4120del site, the FBN1 gene c.6820T>G site, and the FBN1 gene c.7038_7039del site. The probes for the sites are as follows: Targeting the FBN1 gene c.4120del site: FBN1 gene wild-type probe Aw: 5′-ATGCTGGCTGCACATATGGGT-3′; FBN1 gene mutant probe Am: 5'-ATGCTGGCTGCCATATGGGTT-3', Targeting the FBN1 gene c.6820T>G site: FBN1 gene wild-type probe Bw: 5′-TCCACAGATGCACATATATGTG-3′; FBN1 gene mutant probe Bm: 5'-TCCACAGATGCCCATATATGTG-3', Targeting the FBN1 gene c.7038_7039del site: FBN1 gene wild-type probe Cw: 5′-GATCTGACACATGTTTTGTAGC-3′; FBN1 gene mutant probe Cm: 5'-CGATCTGACACATTTTGTAGCA-3'.

Images