Double-tube multiplex fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting osteogenesis insufficiency V-type IFITM5 gene variation site

Abstract

The invention relates to a detection kit for detecting osteogenesis insufficiency V-type IFITM5 gene variation sites based on a TaqMan probe by a double-tube multiple real-time fluorescent quantitative PCR (Polymerase Chain Reaction) technology. The detection kit comprises a PCR primer pair and a PCR primer pair respectively aiming at IFITM5 gene c.-14Cgt, a PCR primer pair and a PCR primer pair respectively aiming at IFITM5 gene c.-14Cgt; t, c.-9 Cgt; a, c.119 Cgt; g and c.119 Cgt; the sequences of the primer and the probe are shown as SEQ ID NO.1-9. The primer, the probe and the kit provided by the invention can realize single detection of multi-site variation, and the primer, the probe and the kit provided by the invention can simultaneously detect four IFITM5 pathogenic variation sites only by one PCR reaction, so that the detection efficiency is remarkably improved, and the detection cost is reduced. Compared with a traditional Sanger sequencing method or a PCR method for single mutation, the method has the advantages that the detection time can be greatly shortened, and the cost is saved.

Description

Technical Field

[0001] The present invention relates to the field of gene diagnosis technology, and particularly relates to a primer and probe composition and a kit for detecting the variant sites of the IFITM5 gene of osteogenesis imperfecta type V. Background Art

[0002] Osteogenesis Imperfecta (OI) is a group of hereditary connective tissue diseases characterized by increased bone fragility, repeated fractures, and skeletal deformities. According to clinical manifestations and genetic etiologies, OI is usually divided into types I to XXII, and the disease severity and inheritance patterns vary among different types. Among them, osteogenesis imperfecta type V is a special type of OI, accounting for about 5% of OI cases, and its clinical manifestations are different from those of traditional OI types. In addition to the typical characteristics of brittle bones and easy fractures, patients with type V OI also have some unique clinical features, including reported hyperplastic callus of the interosseous membrane of the forearm, radial head dislocation, and ectopic ossification of the interosseous membrane between the femur and talus.

[0003] Different from the classic OI types, which are mainly caused by mutations in type I collagen genes (COL1A1, COL1A2), osteogenesis imperfecta type V is mainly caused by mutations in the IFITM5 gene. The IFITM5 (Interferon-Induced Transmembrane Protein 5) gene encodes an interferon-induced transmembrane protein 5. The IFITM5 protein is expressed in osteoblasts and participates in the regulation of the mineralization process of the bone matrix. Mutations in IFITM5 can affect the function of osteoblasts, leading to abnormal bone mineralization and thus causing type V OI.

[0004] Currently, 4 mutations of the IFITM5 gene (NM_001025295.3) have been proven to be pathogenic variants through genetic and functional studies. Among them, the most common IFITM5 mutation is the heterozygous mutation c.-14C>T in the 5' untranslated region (5'UTR) of the gene (PubMed: 22863190). There are also mutations in exon 1, such as c.119C>G and c.119C>T, which have also been reported to be associated with type V OI (PubMed: 24478195, 30985308). The inventors of the present invention discovered a new variant c.-9C>A in the 5'UTR of the IFITM5 gene in neonates with osteogenesis imperfecta and easy fractures, effectively expanding the spectrum of pathogenic variants of the IFITM5 gene. Through morphological studies of mouse models in the present invention, the pathogenicity of the c.-9C>A variant was further demonstrated. The c.-9C>A variant causes disease through the same mechanism as the common c.-14C>T variant and may be a pathogenic variant of the IFITM5 gene unique to the Chinese population. Research data show that almost all type V OI patients are caused by the above 4 IFITM5 gene variants. Therefore, detecting the above-mentioned IFITM5 gene mutation combination can effectively achieve etiological diagnosis and genetic counseling guidance for type V OI patients.

[0005] Currently, there is no commercial kit for detecting pathogenic variants of type V OI. Commonly used methods in scientific research include Sanger sequencing, next-generation sequencing (NGS), and polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). Among them, Sanger sequencing has relatively low throughput, high cost, and long detection time, so it is not suitable for large-scale rapid screening and clinical promotion; NGS can sequence multiple genes or genomic regions simultaneously, but type V OI usually has typical clinical phenotypes, and data analysis is relatively complex. Moreover, for the detection of mutations at known loci, the cost and time advantages are not obvious; for PCR-RFLP, gel electrophoresis of the amplification products is required, which is not only cumbersome to operate, but also non-closed management operations are prone to contamination, resulting in false positive results.

[0006] Therefore, we propose a multiplex fluorescence quantitative PCR primer, probe, kit and its application for the variant sites of the IFITM5 gene in osteogenesis imperfecta type V. Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a primer and probe composition, a kit and a detection method for multiplex fluorescence PCR to detect IFITM5 gene mutations. The kit uses 1 pair of PCR primers, 4 fluorescence channels and 2 reaction wells to achieve the detection of 4 mutations of the IFITM5 gene, specifically including specific primers and TaqMan probe compositions for the c.-14C>T, c.-9C>A, c.119C>G and c.119C>T mutation sites of the IFITM5 gene respectively. The present invention can solve the problem of detecting trace samples, improve the detection qualification rate, simplify the operation, improve the detection throughput and reduce the detection cost at the same time.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The specific primer and probe composition described in the present invention is designed by using Primer 6.0 software. Specifically:

[0010] The PCR primers can amplify the genomic regions containing the c.-14C>T, c.-9C>A, c.119C>G and c.119C>T mutations of the IFITM5 gene. The upstream primer contains a mismatched base modification, which is located at the third position from the bottom of the sequence and is represented by a lowercase letter in the sequence. The specificity of the PCR primer is improved by introducing the mismatched base; the specific sequence is:

[0011] Upstream primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3' (SEQ ID NO.1), downstream primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3' (SEQ ID NO.2);

[0012] 1) The probe for the c.-14C>T site of the IFITM5 gene:

[0013] Wild-type probe Aw: 5'-TTCCAGCGCCGTCTCTTCCAC-3' (SEQ ID NO.3), mutant probe Am: 5'-TTCCAGCGCCATCTCTTCCAC-3' (SEQ ID NO.4)

[0014] 2) The probe for the c.-9C>A site of the IFITM5 gene:

[0015] Wild-type probe Bw: 5'-TCATGGGTTCCAGCGCCGTCTC-3' (SEQ ID NO.5), mutant probe Bm: 5'-CATGGGTTCCATCGCCGTCTC-3' (SEQ ID NO.6)

[0016] 3) Probes for the c.119C>G locus of the IFITM5 gene:

[0017] Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3' (SEQ ID NO.7), mutant probe Cm: 5'-GCTGAACACCCACCAGATCAA-3' (SEQ ID NO.8)

[0018] 4) Probes for the c.119C>T locus of the IFITM5 gene:

[0019] Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3' (SEQ ID NO.7), mutant probe Dm: 5'-GCTGAACACCAACCAGATCAA-3' (SEQ ID NO.9)

[0020] The 5' end of the probe is labeled with different reporter fluorescent groups, and the 3' end is labeled with an MGB quenching fluorescent group.

[0021] In the same reaction system, the 5' ends of the probes are labeled with different reporter fluorescent groups. For example, in reaction system A, the 5' ends of the 4 probes for detecting 2 loci are respectively modified with different recognizable fluorescent dyes, including but not limited to 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, LCRed640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, LC Red705, etc.

[0022] A detection kit for detecting the variant loci of the IFITM5 gene in osteogenesis imperfecta type V by a TaqMan probe-based double-tube multiplex real-time fluorescence quantitative PCR technique, comprising reaction system A and reaction system B;

[0023] The reaction system A includes the following detection primers and detection probes:

[0024] The detection primers:

[0025] Forward primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3',

[0026] Reverse primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3'

[0027] The detection probe:

[0028] For the c.-14C>T locus of the IFITM5 gene:

[0029] Wild-type probe Aw: 5'-TTCCAGCGCCGTCTCTTCCAC-3',

[0030] Mutant probe Am: 5'-TTCCAGCGCCATCTCTTCCAC-3';

[0031] For the c.119C>T locus of the IFITM5 gene:

[0032] Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3',

[0033] Mutant probe Dm: 5'-GCTGAACACCAACCAGATCAA-3';

[0034] The 5' end of the probe is labeled with a reporter fluorophore, and the 3' end is labeled with a quenching fluorophore; in reaction system A, the reporter fluorophores carried by the 5' ends of the probes are different, and can be distinguished from each other by a fluorescence quantitative PCR instrument according to different spectral wavelengths;

[0035] The reporter fluorophore 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 fluorophore is MGB. In the same multiplex reaction system, the 5' ends of the probes are respectively labeled with different reporter fluorophores, and can be distinguished from each other by a fluorescence quantitative PCR instrument according to different spectral wavelengths.

[0036] The reaction system A is 20 μL, and each 20 μL of reaction system A contains:

[0037] 2×premix Taq TM10 μL of buffer, 1 μL of 10 μmoL / L upstream primer, 1 μL of 10 μmoL / L downstream primer, 1 μL of 10 μmoL / L probe Aw, 1 μL of 10 μmoL / L probe Am, 1 μL of 10 μmoL / L probe Cw, 1 μL of 10 μmoL / L probe Dm, 1 μL of template DNA (≥10 ng), 3 μL of RNase- and DNase-free water.

[0038] The reaction system B includes the following detection primers and detection probes:

[0039] The detection primers:

[0040] Upstream primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3',

[0041] Downstream primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3

[0042] The detection probes:

[0043] For the c.-9C>A site of the IFITM5 gene:

[0044] Wild-type probe Bw of the IFITM5 gene: 5'-TCATGGGTTCCAGCGCCGTCTC-3',

[0045] Mutant-type probe Bm of the IFITM5 gene: 5'-CATGGGTTCCATCGCCGTCTC-3';

[0046] For the c.119C>G site of the IFITM5 gene:

[0047] Wild-type probe Cw of the IFITM5 gene: 5'-GCTGAACACCGACCAGATCAA-3',

[0048] Mutant-type probe Cm of the IFITM5 gene: 5'-GCTGAACACCCACCAGATCAA-3';

[0049] The 5' end of the probe is labeled with a reporter fluorophore, and the 3' end is labeled with a quencher fluorophore; in the reaction system B, the reporter fluorophores carried by the 5' ends of the probes are different and can be distinguished from each other by a fluorescence quantitative PCR instrument according to different spectral wavelengths;

[0050] The reporting fluorophore 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, LC Red640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, LC Red705, etc., and the quenching fluorophore is MGB. In the same multiplex reaction system, the 5' ends of the probes are respectively provided with different reporting fluorophores, and can be mutually distinguished by a fluorescence quantitative PCR instrument according to different spectral wavelengths.

[0051] The reaction system B is 20 μL, and each 20 μL of the reaction system B contains:

[0052] 2×premix Taq TM Buffer 10 μL, upstream primer at 10 μmoL / L 1 μL, downstream primer at 10 μmoL / L 1 μL, probe Bw at 10 μmoL / L 1 μL, probe Bm at 10 μmoL / L 1 μL, probe Cw at 10 μmoL / L 1 μL, probe Cm at 10 μmoL / L 1 μL, template DNA 1 μL (≥10 ng), RNase- and DNase-free water 3 μL.

[0053] For the detection kit, when performing double-tube multiplex real-time fluorescence quantitative PCR amplification of the TaqMan probe, the amplification reaction procedure is:

[0054]

[0055] The present invention also provides the application of a reagent for detecting IFITM5 gene mutation sites in the preparation of a detection reagent for osteogenesis imperfecta type V. The IFITM5 gene mutation sites include at least one of the following sites: IFITM5 gene c.-14C>T site, IFITM5 gene c.-9C>A site, IFITM5 gene c.119C>G site, IFITM5 gene c.119C>T site;

[0056] The gene number of the wild-type IFITM5 gene in the NCBI database is: NM_001025295.3;

[0057] Preferably, the c.-14C>T, c.119C>G, and c.119C>T mutations of the IFITM5 gene are pathogenic mutations of the IFITM5 gene proven by functional studies reported in the literature. The c.-9C>A locus was first reported by the inventors and its pathogenic effect was first demonstrated in transgenic mice.

[0058] The present invention also provides the use of a reagent for detecting a mutation site of the IFITM5 gene in the preparation of a detection kit for osteogenesis imperfecta type V. The reagent for detecting a mutation site of the IFITM5 gene is one or more of a probe and a primer for detecting a mutation site of the IFITM5 gene.

[0059] The primers include primers for the c.-14C>T, c.-9C>A, c.119C>G, and c.119C>T mutation sites of the IFITM5 gene, specifically as follows:

[0060] Forward primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3' (SEQ ID NO.1)

[0061] Reverse primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3' (SEQ ID NO.2).

[0062] The probes include probes for at least one of the c.-14C>T site of the IFITM5 gene, the c.-9C>A site of the IFITM5 gene, the c.119C>G site of the IFITM5 gene, and the c.119C>T site of the IFITM5 gene. The probes for the above sites are as follows:

[0063] The probe for the c.-14C>T site of the IFITM5 gene:

[0064] Wild-type probe Aw: 5'-TTCCAGCGCCGTCTCTTCCAC-3' (SEQ ID NO.3)

[0065] Mutant probe Am: 5'-TTCCAGCGCCATCTCTTCCAC-3' (SEQ ID NO.4). The probe for the c.-9C>A site of the IFITM5 gene:

[0066] Wild-type probe Bw: 5'-TCATGGGTTCCAGCGCCGTCTC-3' (SEQ ID NO.5)

[0067] Mutant probe Bm: 5'-CATGGGTTCCATCGCCGTCTC-3' (SEQ ID NO.6). The probe for the c.119C>G site of the IFITM5 gene:

[0068] Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3' (SEQ ID NO.7),

[0069] Mutant probe Cm: 5'-GCTGAACACCCACCAGATCAA-3' (SEQ ID NO.8), the probe for the c.119C>T locus of the IFITM5 gene:

[0070] Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3' (SEQ ID NO.7),

[0071] Mutant probe Dm: 5'-GCTGAACACCAACCAGATCAA-3' (SEQ ID NO.9). Compared with the prior art, the advantages of the present invention are as follows:

[0072] 1) Detection of multiple-site mutations in a single test: The primers, probes and kits provided by the present invention can detect four pathogenic mutation sites of IFITM5 simultaneously with only one PCR reaction, significantly improving the detection efficiency. Compared with traditional Sanger sequencing or PCR methods targeting a single mutation, the detection time can be greatly shortened and costs can be saved.

[0073] 2) Rapid diagnosis: The multiplex PCR reaction is fast, and combined with the real-time fluorescence detection, it can achieve rapid diagnosis of type V OI, which is crucial for early clinical diagnosis and timely treatment.

[0074] 3) Economical and efficient: Compared with high-throughput sequencing technologies such as NGS, the multiplex fluorescence quantitative PCR method has lower costs, simpler operation, and is more suitable for clinical routine detection and application in primary medical institutions.

[0075] 4) Targeting known pathogenic mutations: The primers, probes and kits provided by the present invention focus on detecting known and high-frequency pathogenic mutations, with strong targeting and high clinical application value. For the diagnosis of type V OI, detecting these four mutation sites can cover almost all cases, thereby improving the accuracy and efficiency of diagnosis.

[0076] 5) Reliable detection performance: The primers, probes and kits provided by the present invention all reach 100% in performance indicators such as coincidence rate, sensitivity and specificity. Description of the Drawings

[0077] Figure 1 It is the skeletal CT imaging result of the transgenic mouse at the c.-9C>A locus of the IFITM5 gene in Example 1. In the figure: A is the femur, B is the skull, C is the forelimb bone, and D is the rib.

[0078] Figure 2 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe at the c.-14C>T locus of the IFITM5 gene in Example 3. Among them, the left diagram is the amplification curve result diagram of the c.-14C wild type, and the right diagram is the amplification curve result diagram of the heterozygote at the c.-14C>T locus of the IFITM5 gene.

[0079] Figure 3 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe at the c.-9C>A locus of the IFITM5 gene in Example 3. Among them, the left diagram is the amplification curve result diagram of the c.-9C wild type, and the right diagram is the amplification curve result diagram of the heterozygote at the c.-9C>A locus of the IFITM5 gene.

[0080] Figure 4 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe at the c.119C>G locus of the IFITM5 gene in Example 3; among them, the right diagram is the amplification curve result diagram of the heterozygote at the c.119C>G locus of the IFITM5 gene; the left diagram is the amplification curve result diagram of the c.119C wild type.

[0081] Figure 5 It is the real-time fluorescence quantitative PCR result diagram of the TaqMan probe at the c.119C>T locus of the IFITM5 gene in Example 3; among them, the right diagram is the amplification curve result diagram of the heterozygote at the c.119C>T locus of the IFITM5 gene; the left diagram is the amplification curve result diagram of the c.119C wild type.

[0082] Figure 6 It is the Sanger sequencing result diagram of the c.-14C locus of the IFITM5 gene in Example 7, where the red square is the detection site.

[0083] Figure 7 It is the Sanger sequencing result diagram of the c.-9C locus of the IFITM5 gene in Example 7, where the red square is the detection site.

[0084] Figure 8 It is the Sanger sequencing result diagram of the c.119C locus of the IFITM5 gene in Example 7; among them, the red square is the detection site. Detailed implementation manners

[0085] The following is a detailed description of the content of the present invention in conjunction with the specification drawings and examples:

[0086] Example 1 Transgenic mouse research proves the pathogenicity of the c.-9C>A variation of the IFITM5 gene

[0087] In this example, the pathogenicity of the c.-9C>A variant of osteogenesis imperfecta type V was demonstrated through a morphological mechanism study based on a transgenic mouse model. This variant was discovered by the inventors in a neonatal patient with a transverse fracture of the clavicle and a slightly delayed early healing process. Through multi-gene next-generation sequencing (NGS) technology, a candidate pathogenic variant of type V OI in the IFITM5 gene was identified. This variant was a de novo mutation not carried by the patient's parents. c.-9C>A is located in the 5’UTR of the IFITM5 gene. Bioinformatics prediction and further in vitro functional studies of cells and proteins confirmed that this variant could generate a new in-frame start codon 9 bp upstream of the original start codon, resulting in the mutant product (MEP-IFITM5) having three additional amino acid residues (Met-Glu-Pro). Comparing the newly discovered IFITM5 mutant protein (c.-9C>A, MEP-IFITM5) with the common mutant protein (c.-14C>T, MALEP-IFITM5), we found that they differed by only two amino acid residues (HGMD ID: CR2013941). Since the patient was young and did not show typical clinical phenotypes of osteogenesis imperfecta type V, the pathogenicity rating of this variant was of uncertain significance according to the ACMG guidelines.

[0088] The inventors created a transgenic mouse (MEP-IFITM5) with a mutant IFITM5 controlled by the osteoblast-specific Col1a1 2.3 kb promoter in Example 1 to analyze the effect of the new variant on bone development and clarify the pathogenicity of the c.-9C>A variant in the IFITM5 gene. The detailed technical process includes:

[0089] 1) Construction of transgenic mice

[0090] (1) Clone the Col1a1 2.3 kb promoter sequence and construct a genetic engineering vector plasmid containing this promoter.

[0091] (2) Clone the cDNA of murine IFITM5 and clone it downstream of the Col1a1 2.3 kb promoter.

[0092] (3) Use a gene mutagenesis kit for mutagenesis to obtain the c.-9C>A mutant by site-directed mutagenesis.

[0093] The mutagenic primer sequences used are as follows: 5'-accagtctgagtgtggaagagacggcg A tggaacccatg3' (SEQ ID NO.10)

[0094] (4) Construction of mutant mice: The construction of mutant mice was assisted by Fuzhou Furui Medical Laboratory Co., Ltd. and the Animal Center of the Comparative Medicine Department of the 900th Hospital. The mutant and wild-type IFITM5 constructs were injected to construct mutant mice, maintaining the FVB / N background of the mice.

[0095] (5) Morphological observation: Record the activity and mortality of the mice; measure the development indexes such as the limb length and the head-to-tail length of the mice.

[0096] 2) X-ray imaging analysis of the skeletal development of transgenic mice and wild mice

[0097] The X-ray films of transgenic animals and their respective non-transgenic littermates were taken using a sample radioanalysis system. The specific steps were as follows: The mutant transgenic mice and their non-transgenic littermates or wild-type controls were sacrificed at E18.5 or E15.5, fixed with 95% ethanol, soaked in alcian blue overnight, and soaked in 2% KOH solution for 24 hours to remove the residual soft tissues. The bone preparations were stained with alizarin red solution overnight, washed with 1% KOH / 20% glycerol solution, and stored in a 1:1 solution of glycerol / 95% ethanol.

[0098] The results of the morphological study of the mice found that a total of 12 chimeric transgenic mice were constructed in this example, of which 8 died. The c.-9C>A mutation frequency in the dead group of mice was significantly higher than that in the surviving group (63.4% vs 35.3%). Histological analysis of the heart and liver tissues did not find any abnormalities, suggesting that the c.-9C>A mutant showed a perinatal lethal phenotype with embryonic lethality. CT observation showed that the long bones and ribs of the mutant mice were deformed, and the femur of the hind limbs was bent ( Figure 1 A), the cranial sutures were widened ( Figure 1 B), the radius and ulna of the forelimbs were also severely bent at a single site ( Figure 1 C), and the ribs were thin and bent ( Figure 1 D), suggesting that the c.-9C>A variation could lead to severe abnormal skeletal development in mice. The above results of the morphological study of the mice showed that the c.-9C>A variation of the IFITM5 gene could lead to the phenotype of osteogenesis imperfecta, and for the first time, the pathogenicity of this variation was demonstrated through an animal model.

[0099] Example 2 Primer and probe compositions and kits for detecting the variant sites of the IFITM5 gene in osteogenesis imperfecta type V

[0100] (1) The primer and probe compositions and kits for detecting the variant sites of the IFITM5 gene in osteogenesis imperfecta type V are used for screening or diagnosing osteogenesis imperfecta type V; including specific primers and TaqMan probe compositions respectively targeting the c.-14C>T, c.-9C>A, c.119C>G, and c.119C>T variant sites of the IFITM5 gene.

[0101] The specific primer and probe composition described above was designed using Primer 6.0 software. Specifically:

[0102] The primers include primers targeting the c.-14C>T, c.-9C>A, c.119C>G, and c.119C>T mutation sites of the IFITM5 gene. Specifically:

[0103] Forward primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3',

[0104] Reverse primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3'.

[0105] The probes include probes targeting at least one of the c.-14C>T site of the IFITM5 gene, the c.-9C>A site of the IFITM5 gene, the c.119C>G site of the IFITM5 gene, and the c.119C>T site of the IFITM5 gene. The probes for the respective sites are as follows:

[0106] The probe for the c.-14C>T site of the IFITM5 gene:

[0107] Wild-type probe Aw: 5'-TTCCAGCGCCGTCTCTTCCAC-3',

[0108] Mutant probe Am: 5'-TTCCAGCGCCATCTCTTCCAC-3

[0109] The probe for the c.-9C>A site of the IFITM5 gene:

[0110] Wild-type probe Bw: 5'-TCATGGGTTCCAGCGCCGTCTC-3',

[0111] Mutant probe Bm: 5'-CATGGGTTCCATCGCCGTCTC-3

[0112] The probe for the c.119C>G site of the IFITM5 gene:

[0113] Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3',

[0114] Mutant probe Cm: 5'-GCTGAACACCCACCAGATCAA-3

[0115] The probe for the c.119C>T site of the IFITM5 gene:

[0116] Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3',

[0117] Mutant probe Dm: 5'-GCTGAACACCAACCAGATCAA-3'.

[0118] The 5'-end of the probe is attached with different reporter fluorescent groups, and the 3'-end is attached with an MGB quenching fluorescent group.

[0119] In the reaction system, the 5'-ends of the probes are respectively attached with different reporter fluorescent groups. For example, in reaction system A, the 5'-ends of 4 probes for detecting 2 sites are respectively modified with different recognizable fluorescent dyes, including but not limited to 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, LCRed640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, LC Red705, etc.

[0120] (2) Construction of the multiplex reaction system scheme: To determine the feasibility of constructing a multiplex reaction system with the above probes, the present invention evaluates the possible interactions between the probes through an interference experiment. Construct a quantitative reaction system for a single site and two-by-two sites according to the following reaction system: 2×premix Taq TM Buffer (Tris-HCl, pH 8.9; 20 mM KCl; 100 mM MgCl2 3 mM, BaoRiYuan Biotechnology (Beijing) Co., Ltd.) 10 μL, upstream primer (10 μmoL) 1 μL, downstream primer (10 μmoL) 1 μL, DNA 1 μL, wild-type (w) TaqMan probe (10 μmoL) 1 μL, mutant (m) TaqMan probe (10 μmoL) 1 μL, add ddH2O to make up to 20 μL. Record the Ct value results of each reaction system. If there is no statistical difference (p > 0.05) between the Ct values of the two-by-two site reaction system and the single site reaction system, then there is no mutual influence between the probes in this duplex system; conversely, if there is a statistical difference (p ≤ 0.05), then there is mutual interference between the probes in this duplex system. The specific results are shown in the following table: There is an interaction between probe A and probe B, and between probe C and probe D. Determine that reaction system A includes probe A and probe D; reaction system B includes probe B and probe C.

[0121]

[0122] (3) The reagent components of the kit can use common PCR amplification reaction reagents, including buffer, ions, dNTP, PCR polymerase, water or other PCR additives, including but not limited to 2×premix Taq used in this embodiment TM Buffer (LA Taq TM Version 2.0, Baoruiyi Biotechnology (Beijing) Co., Ltd.) and RNase- and DNase-free water

[0123] (4) The kit adopts a double-tube multiplex fluorescence PCR reaction system, specifically as follows:

[0124] Reaction system A: c.-14C>T locus and c.119C>T locus of the IFITM5 gene

[0125] <![CDATA[2×premix Taq TM Buffer]]> 10 μL Forward primer (10 μmoL) 1 μL Reverse primer (10 μmoL) 1 μL TaqMan probe (10 μmoL) 4 μL (1 μL each of probe Aw, Cw; probe Am, Dm) Template DNA 1 μL (≥10 ng) RNase- and DNase-free water 3 μL Total volume 20 μL

[0126] Reaction system B: c.-9C>A locus and c.119C>G locus of the IFITM5 gene

[0127] <![CDATA[2×premix Taq TM Buffer]]> 10 μL Forward primer (10 μmoL) 1 μL Reverse primer (10 μmoL) 1 μL TaqMan probe (10 μmoL) 4 μL (1 μL each of probe Bw, Cw; probe Bm, Cm) Template DNA 1 μL (≥10 ng) RNase- and DNase-free water 3 μL Total volume 20 μL

[0128] The amplification reaction procedure is as follows:

[0129]

[0130] (5) Operating procedure for the multiplex probe reagent to detect the c.-14C>T, c.-9C>A, c.119C>G and c.119C>T mutation sites of the IFITM5 gene in osteogenesis imperfecta type V:

[0131] Collect clinical tissue samples of the proband or family members of osteogenesis imperfecta type V, including but not limited to peripheral blood anticoagulated with EDTA / sodium citrate

[0132] Extract genomic DNA from the clinical tissue samples and perform quality control. Measure the purity of the DNA using Nanodrop one. The OD260nm / OD280nm of the obtained genomic DNA is between 1.7 and 2.0. Measure the concentration of the DNA using Nanodrop one. The concentration of the obtained genomic DNA is ≥50 ng / μL, and the total amount is ≥5 μg. Store at -20°C

[0133] Using the genomic DNA sample, prepare a double-tube multiplex fluorescence PCR reaction system and perform an amplification reaction on a fluorescence quantitative PCR instrument according to the above reaction procedure

[0134] Result interpretation: After the fluorescence quantitative PCR is completed, data analysis and processing are performed using software, and the results of each subject can be obtained. By judging whether there are amplification peaks of wild-type and mutant specific probes at each detection site of the subject ( Figures 2 - 5 ), the ΔRn threshold is automatically judged by the fluorescence quantitative PCR instrument. The results are judged according to the following criteria to determine whether the subject carries the pathogenic sites and genotypes of the IFITM5 gene.

[0135]

[0136] Results of the performance study of the kit for detecting IFITM5 gene variant sites in osteogenesis imperfecta type V

[0137] (1) Analytical performance study protocol

[0138] Clinical sample collection: To study the analytical performance of the duplex multiplex fluorescence TaqMan probe PCR detection kit for IFITM5 gene variant sites in osteogenesis imperfecta type V of the present invention, 60 clinical samples were used as the research objects for performance study in this example. The relevant samples are stored by the 900th Hospital of the Chinese People's Liberation Army and Furi Medical Laboratory, including: 30 cases of positive cases of genetic diagnosis of osteogenesis imperfecta (including 13 cases of positive samples with pathogenic variants in the COL1A1 gene, 13 cases of positive samples with pathogenic variants in the COL1A2 gene, and 1 case each of positive samples with variant sites of c.-14C>T, c.-9C>A, c.119C>G, and c.119C>T in the IFITM5 gene), and 30 normal controls without clinical skeletal system abnormalities.

[0139] (2) Performance verification protocol: The detection results of all samples were compared with the NGS analysis data and / or Sanger sequencing verification results. The consistency, sensitivity, and specificity of the detection of the c.-14C>T, c.-9C>A, and c.119C>G variant sites of the IFITM5 gene in osteogenesis imperfecta type V by the kit of the present invention were calculated respectively.

[0140] The consistency rate was compared by parallel detection of clinical samples with the reference method. The reference methods include but are not limited to: the gold standard method, industry-recognized methods, and methods whose performance has been verified to meet the requirements and meet the clinical intended use (such as: the same detection method used by laboratories accredited by ISO15189). In this study, the gold standard method (Sanger) and the industry-recognized method (high-depth NGS sequencing) were selected for comparison with the duplex multiplex fluorescence TaqMan probe PCR detection method of the present invention, and the detection results were recorded and tabulated as follows:

[0141]

[0142] The calculation formulas are as follows:

[0143] Coincidence rate = (a + d) / (a + b + c + d) × 100%

[0144] Sensitivity = a / (a + c) × 100%

[0145] Specificity = d / (b + d) × 100%

[0146] (3) The system and steps for Sanger sequencing verification:

[0147] The PCR amplification systems (20 μl) for the c.-14C>T, c.-9C>A, c.119C>G, and c.119C>T mutation sites are respectively: 2×premix Taq TM Buffer 10 μL, upstream primer (10 μmoL) 1 μL, downstream primer (10 μmoL) 1 μL, ddH2O 6 μL, DNA 2 μL; PCR reaction program: 95°C for 5 min, 35 cycles (95°C for 30 s, 64°C for 1 min), 72°C for 10 min, and stored at 4°C. After PCR amplification, 1% agarose gel electrophoresis was used for detection. After cutting the gel, it was recovered with the 《Common Agarose Gel DNA Recovery Kit (DP209)》, and the product was diluted to 10 ng / μL, and the recovered product was purified with Taq enzyme. All PCR products were subjected to Sanger sequencing analysis on the ABI 3730XL (Applied Biosystems) platform using the amplification primers, and the sequencing reaction was commissioned to Fuzhou Furi Medical Laboratory Co., Ltd. to complete.

[0148] (4) Results of the analysis performance study

[0149] The coincidence rate of the kit for detecting the c.-14C>T mutation site of the IFITM5 gene in osteogenesis imperfecta type V of the present invention with the gold standard or industry-recognized method is 100%, and its detection sensitivity and specificity are both 100%. For specific results, see the following table and the Sanger sequencing verification results ( Figure 6 ):

[0150]

[0151]

[0152] The coincidence rate of the kit for detecting the c.-9C>A mutation site of the IFITM5 gene in osteogenesis imperfecta type V of the present invention with the gold standard or industry-recognized method is 100%, and its detection sensitivity and specificity are both 100%. For specific results, see the following table and the Sanger sequencing verification results ( Figure 7 ):

[0153]

[0154] The coincidence rate of the kit for detecting the variant site of the IFITM5 gene in osteogenesis imperfecta type V of the present invention for detecting the c.119C>G variant site with the gold standard or industry-recognized method is 100%, and its detection sensitivity and specificity are both 100%. The specific results are shown in the following table and the Sanger sequencing verification results ( Figure 8 ):

[0155]

[0156] The coincidence rate of the kit for detecting the variant site of the IFITM5 gene in osteogenesis imperfecta type V of the present invention for detecting the c.119C>T variant site with the gold standard or industry-recognized method is 100%, and its detection sensitivity and specificity are both 100%. The specific results are shown in the following table and the Sanger sequencing verification results ( Figure 8 ):

[0157]

[0158] The above describes the embodiments of this example, but this example is not limited to the above specific implementation manners. The above specific implementation manners are only illustrative and not restrictive. Under the inspiration of this example, those of ordinary skill in the art can also make many forms, all of which fall within the protection scope of this example.

Claims

1. A detection kit for detecting the variation site of the IFITM5 gene in osteogenesis imperfecta type V by a double-tube multiplex real-time fluorescence quantitative PCR technology based on TaqMan probes, characterized in that: It includes reaction system A and reaction system B; The reaction system A includes the detection primers and detection probes shown below: Detection primers: Forward primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3', Reverse primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3' Detection probes: For the c.-14C>T site of the IFITM5 gene: Wild-type probe Aw: 5'-TTCCAGCGCCGTCTCTTCCAC-3', Mutant probe Am: 5'-TTCCAGCGCCATCTCTTCCAC-3'; For the c.119C>T site of the IFITM5 gene: Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3', Mutant probe Dm: 5'-GCTGAACACCAACCAGATCAA-3'; The 5'-end of the probe is labeled with a reporter fluorophore, and the 3'-end is labeled with a quencher fluorophore; in reaction system A, the reporter fluorophores carried by the 5'-ends of the probes are different; The reaction system B includes the detection primers and detection probes shown below: Detection primers: Forward primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3', Reverse primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3' Detection probes: For the c.-9C>A site of the IFITM5 gene: Wild-type probe Bw of the IFITM5 gene: 5'-TCATGGGTTCCAGCGCCGTCTC-3', Mutant probe Bm of the IFITM5 gene: 5'-CATGGGTTCCATCGCCGTCTC-3'; For the c.119C>G site of the IFITM5 gene: Wild-type probe Cw of the IFITM5 gene: 5'-GCTGAACACCGACCAGATCAA-3', Mutant probe Cm of the IFITM5 gene: 5'-GCTGAACACCCACCAGATCAA-3'; The 5'-end of the probe is labeled with a reporter fluorophore, and the 3'-end is labeled with a quencher fluorophore; in reaction system B, the reporter fluorophores carried by the 5'-ends of the probes are different.

2. The detection kit according to claim 1, wherein: The reaction system A is 20 μL, and each 20 μL of reaction system A contains: 2×premix Taq TM 10 μL of buffer, 1 μL of 10 μmoL / L upstream primer, 1 μL of 10 μmoL / L downstream primer, 1 μL of 10 μmoL / L probe Aw, 1 μL of 10 μmoL / L probe Am, 1 μL of 10 μmoL / L probe Cw, 1 μL of 10 μmoL / L probe Dm, 1 μL of template DNA (≥10 ng), 3 μL of RNase- and DNase-free water.

3. The detection kit according to claim 1, wherein: The reaction system B is 20 μL, and each 20 μL of reaction system B contains: 2×premix Taq TM 10 μL of buffer, 1 μL of 10 μmoL / L upstream primer, 1 μL of 10 μmoL / L downstream primer, 1 μL of 10 μmoL / L probe Bw, 1 μL of 10 μmoL / L probe Bm, 1 μL of 10 μmoL / L probe Cw, 1 μL of 10 μmoL / L probe Cm, 1 μL of template DNA (≥10 ng), 3 μL of RNase- and DNase-free water.

4. The detection kit according to claim 1, wherein: The reported fluorophore 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, LC Red640, Snarf 1, Acid Fuchsin, Cy5.5, LC Red670, LC Red705, and the quenching fluorophore is MGB.

5. The detection kit according to claim 1, characterized in that: When performing duplex multiplex real-time fluorescence quantitative PCR amplification of its TaqMan probe, the amplification reaction program is as follows:

6. Use of a reagent for detecting IFITM5 gene mutation sites in the preparation of a detection kit for osteogenesis imperfecta type V, characterized in that: The reagent for detecting the mutation site of the IFITM5 gene is a probe and primer for detecting the mutation site of the IFITM5 gene; The primers include primers targeting the c.-14C>T, c.-9C>A, c.119C>G, and c.119C>T sites of the IFITM5 gene, specifically: Forward primer: 5'-CTCCCAGATGGGATGTCTGTgAG-3', Reverse primer: 5'-TTCCCGGCCATACTGATAGCTGTG-3'. The probes include probes targeting the c.-14C>T site of the IFITM5 gene, the c.-9C>A site of the IFITM5 gene, the c.119C>G site of the IFITM5 gene, and the c.119C>T site of the IFITM5 gene. The probes for these sites are as follows: Probe targeting the c.-14C>T site of the IFITM5 gene: Wild-type probe Aw: 5'-TTCCAGCGCCGTCTCTTCCAC-3', Mutant probe Am: 5'-TTCCAGCGCCATCTCTTCCAC-3', Probe targeting the c.-9C>A site of the IFITM5 gene: Wild-type probe Bw: 5'-TCATGGGTTCCAGCGCCGTCTC-3', Mutant probe Bm: 5'-CATGGGTTCCATCGCCGTCTC-3', Probe targeting the c.119C>G site of the IFITM5 gene: Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3', Mutant probe Cm: 5'-GCTGAACACCCACCAGATCAA-3', Probe targeting the c.119C>T site of the IFITM5 gene: Wild-type probe Cw: 5'-GCTGAACACCGACCAGATCAA-3', Mutant probe Dm: 5'-GCTGAACACCAACCAGATCAA-3'.

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