Capture probe, familial chylomicronemia syndrome detection kit containing same, and use

Abstract

The present application discloses a capture probe, a familial chylomicronemia syndrome (FCS) detection kit containing same, and a use. The present application attempts to detect FCS-related gene mutations by means of the product and use, thereby providing a clinical detection method for FCS.

Description

Capture probe, familial chylomicronemia syndrome detection kit containing same, and application thereof Technical Field

[0001] The present application relates to the field of biomedical detection, and in particular to a capture probe, a familial chylomicronemia syndrome detection kit containing the same, and applications thereof. Background Art

[0002] Familial chylomicronemia syndrome (FCS) is an autosomal recessive disorder also known as primary hyperlipoproteinemia type I. FCS is characterized by severe fasting hypertriglyceridemia secondary to the accumulation of triglyceride (TG)-rich lipoproteins, particularly chylomicrons, requiring long-term dietary fat restriction. The clinical manifestations of FCS are typically eruptive xanthomas, which are commonly seen on the buttocks, thighs, arms, back, and face, and may also involve the oral mucosa. Other symptoms include epigastric pain, which may be caused by tension in the hepatosplenic capsule, hepatosplenomegaly, and pancreatitis. This rare and nonspecific disorder carries potentially serious clinical consequences, with each affected sibling having a 25% chance of developing the condition.

[0003] Current diagnostic criteria for FCS, primarily derived from European clinical practice, are comprehensive and rely primarily on factors such as triglyceride levels, the patient's response to triglyceride-lowering medications, and a history of frequent abdominal pain or pancreatitis. Conventional laboratory and clinical examinations cannot accurately identify and localize the cause of the disease. Furthermore, changes in triglyceride levels are also seen in the pathogenesis of other diseases and are not entirely specific to FCS. Consequently, FCS is often missed in clinical practice. Overall, current diagnostic methods are unable to provide accurate and timely FCS diagnoses, hindering timely treatment and making accurate identification and localization of FCS challenging. Summary of the Invention

[0004] Based on this, an object of one or more embodiments of the present application includes providing a capture probe that can be used for accurate detection of FCS-related gene mutations.

[0005] In one or more embodiments of the present application, a capture probe is provided, which includes one or more groups of probe combinations 1 to 6;

[0006] Probe combination 1 includes one or more of probes 1 to 26;

[0007] Probe combination 2 includes one or more of probes 27 to 64;

[0008] Probe combination 3 includes one or more probes 65 to 112;

[0009] Probe combination 4 includes one or more of probes 113 to 139;

[0010] Probe combination 5 includes one or more of probes 140 to 382;

[0011] Probe combination 6 includes one or more of probes 383 to 425;

[0012] The probes 1 to 425 are probes constructed for target gene fragments between different start sites and end sites on the following chromosomes and are complementary to the target gene fragments:

[0013] The reference genome is Hg19.

[0014] In some specific embodiments of the present application, the capture probes include multiple groups of probe combinations 1 to 6;

[0015] The probe combination 1 includes multiple probes from probe 1 to probe 26;

[0016] The probe combination 2 includes multiple probes from probes 27 to probes 64;

[0017] The probe combination 3 includes multiple probes from probe 65 to probe 112;

[0018] The probe combination 4 includes multiple probes from probes 113 to probes 139;

[0019] The probe combination 5 includes multiple probes from probes 140 to probes 382;

[0020] The probe combination 6 includes multiple probes from probes 383 to probes 425 .

[0021] In some specific embodiments of the present application, the capture probes include probe combination 1 to probe combination 6;

[0022] The probe combination 1 includes probes 1 to 26;

[0023] The probe combination 2 includes probes 27 to 64;

[0024] The probe combination 3 includes probes 65 to 112;

[0025] The probe combination 4 includes the probes 113 to 139;

[0026] The probe combination 5 includes the probes 140 to 382;

[0027] The probe combination 6 includes probes 383 to 425.

[0028] In another or more embodiments of the present application, there is provided use of the capture probe in preparing a familial chylomicronemia syndrome detection kit.

[0029] In still another one or more embodiments of the present application, a familial chylomicronemia syndrome detection kit is provided, which includes the capture probe described above.

[0030] In some specific embodiments of the present application, the familial chylomicronemia syndrome detection kit further includes one or more of nucleic acid extraction reagents, DNA library construction reagents, sample and library quantification reagents, fragment quality control reagents, hybridization capture reagents, nucleic acid purification reagents, target gene fragment amplification reagents and sequencing reagents.

[0031] In yet another one or more embodiments of the present application, there is provided use of the capture probe in constructing a library for detecting gene mutations associated with familial chylomicronemia syndrome.

[0032] In one or more embodiments of the present application, there is provided use of the detection kit in constructing a library for detecting gene mutations associated with familial chylomicronemia syndrome.

[0033] In one or more embodiments of the present application, a method for detecting a familial chylomicronemia syndrome-related gene mutation is provided, comprising the steps of using the capture probe or the detection kit to capture a target gene fragment containing a familial chylomicronemia syndrome-related gene mutation, and detecting the captured fragment.

[0034] In some specific embodiments of the present application, sequencing is used to detect the captured fragments.

[0035] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. DETAILED DESCRIPTION

[0036] Below in conjunction with embodiment and example, the present invention is described in further detail.Should be understood that these embodiment and example are only used to illustrate the present invention and are not used to limit the scope of the invention, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the present invention more thorough and comprehensive.It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the present invention, and it should be understood that the present invention can be implemented without one or more of these details.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing embodiments and examples and are not intended to limit the present invention.

[0038] the term

[0039] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0040] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0041] In the present invention, "plurality", "multiple", "multiple times", "multiple", etc., unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0042] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0043] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0044] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0045] In the present invention, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention.

[0046] In the present invention, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no conflicts or constraints.

[0047] In the present invention, the terms "first," "second," "third," and "fourth," etc., in "the first aspect," "the second aspect," "the third aspect," and "the fourth aspect," etc., are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," "third," and "fourth," etc., serve only as non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0048] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0049] In the present invention, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0050] The temperature parameters in the present invention, unless otherwise specified, may be either constant temperature or fluctuating within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the precision range of the instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0051] In the present invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0052] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0053] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made according to experimental manuals or conventional conditions in the art, or according to conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0054] FCS is caused by one or more gene mutations that can lead to loss of function in lipoprotein lipase (LPL), apolipoprotein CII (APOC2), lipase maturation factor 1 (LMF1), glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), apolipoprotein AV (APOA5), and glycerol-3-phosphate dehydrogenase 1 (GPD1), thereby affecting chylomicron lipolysis and clearance. In one embodiment of the present application, six genes closely related to FCS (LPL, APOC2, LMF1, GPIHBP1, APOA5, and GPD1) were selected, and a probe panel covering the exons and hotspot intron regions of these six genes was designed for capture sequencing. The aim is to stably and accurately detect FCS-related gene mutations, providing a simpler and more accurate FCS detection method for clinical use. This is of great significance for the clinical diagnosis and treatment of FCS.

[0055] In the first aspect of the embodiment of the present application

[0056] The present application provides a capture probe, which includes one or more groups of probe combinations 1 to 6;

[0057] Probe combination 1 includes one or more of probes 1 to 26;

[0058] Probe combination 2 includes one or more of probes 27 to 64;

[0059] Probe combination 3 includes one or more probes 65 to 112;

[0060] Probe combination 4 includes one or more of probes 113 to 139;

[0061] Probe combination 5 includes one or more of probes 140 to 382;

[0062] Probe combination 6 includes one or more of probes 383 to 425;

[0063] The probes 1 to 425 are probes constructed for target gene fragments between different start sites and end sites on the following chromosomes and are complementary to the target gene fragments:

[0064] The reference genome is Hg19.

[0065] The capture probes provided in the embodiments of the present application can be used to cover the exons and hotspot intron regions of six genes (LPL, APOC2, LMF1, GPIHBP1, APOA5, and GPD1) that are closely related to FCS. It should be understood that, according to the needs, some of the probe combinations can be selected to detect some target genes, for example, any one, two, three, four, or five groups selected from probe combination 1 to probe combination 6. In order to meet the detection requirements of some regions of the target gene, a corresponding number of probes can be selected from the corresponding probe combination, for example, 1, 2, 3...24, 25, or 26 probes in probe combination 1 can be selected.

[0066] Optionally, the capture probes include multiple groups of probe combinations 1 to 6;

[0067] The probe combination 1 includes multiple probes from probe 1 to probe 26;

[0068] The probe combination 2 includes multiple probes from probes 27 to probes 64;

[0069] The probe combination 3 includes multiple probes from probe 65 to probe 112;

[0070] The probe combination 4 includes multiple probes from probes 113 to probes 139;

[0071] The probe combination 5 includes multiple probes from probes 140 to probes 382;

[0072] The probe combination 6 includes multiple probes from probes 383 to probes 425 .

[0073] Further optionally, the capture probes include probe combination 1 to probe combination 6;

[0074] The probe combination 1 includes probes 1 to 26;

[0075] The probe combination 2 includes probes 27 to 64;

[0076] The probe combination 3 includes probes 65 to 112;

[0077] The probe combination 4 includes the probes 113 to 139;

[0078] The probe combination 5 includes the probes 140 to 382;

[0079] The probe combination 6 includes probes 383 to 425.

[0080] The second aspect of the embodiment of the present application

[0081] The examples of the present application provide the use of the capture probe in preparing a familial chylomicronemia syndrome detection kit.

[0082] The third aspect of the embodiments of the present application

[0083] The embodiment of the present application provides a familial chylomicronemia syndrome detection kit, which includes the capture probe described above.

[0084] Optionally, the familial chylomicronemia syndrome detection kit further includes one or more of nucleic acid extraction reagents, DNA library construction reagents, sample and library quantification reagents, fragment quality control reagents, hybridization capture reagents, nucleic acid purification reagents, target gene fragment amplification reagents and sequencing reagents.

[0085] The fourth aspect of the embodiments of the present application

[0086] The examples of the present application provide the use of the capture probe in constructing a library for detecting gene mutations associated with familial chylomicronemia syndrome.

[0087] The fifth aspect of the embodiment of the present application

[0088] The present application provides an example of using the detection kit in constructing a familial chylomicronemia syndrome-related gene mutation detection library.

[0089] The sixth aspect of the embodiments of the present application

[0090] The embodiment of the present application provides a method for detecting a familial chylomicronemia syndrome-related gene mutation, which includes using the capture probe or the detection kit to capture a target gene fragment containing a familial chylomicronemia syndrome-related gene mutation, and detecting the captured fragment.

[0091] Optionally, the captured fragments are detected by sequencing.

[0092] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0093] The reagents used in the detection process of this embodiment are listed as follows:

[0094] Table 1

[0095] The equipment used in the detection process of this embodiment is listed as follows:

[0096] Table 2

[0097] Example 1

[0098] The capture sequencing detection process of the six FCS-related genes in this example is divided into four steps: sample preparation, library construction, target region capture and sequencing.

[0099] 1.Panel design

[0100] In this example, the capture probe panel for 16 FCS-associated genes, including LPL, APOC2, LMF1, GPIHBP1, APOA5, and GPD, covers an 18.6 kb region, encompassing all exons and hotspot introns of the six genes closely associated with FCS (Table 3). The high-density probe design ensures that each target region is covered by at least two probes (see Table 4).

[0101] Table 3. Probe coverage of 6 FCS-related genes

[0102] Table 4. Capture probes

[0103] This example uses target capture probes that fully cover all exons and hotspot introns of the six genes mentioned above, ensuring accurate and efficient detection of FCS-related gene mutations, providing a basis for clinical diagnosis and medication guidance for FCS. The nucleotide sequences of probes 1 to 425 are shown in SEQ ID NOs. 1 to 425.

[0104] 2. Experimental Design

[0105] The detection experimental process includes four main steps: sample preparation, library construction, target region enrichment, and sequencing. First, about 300 ng of gDNA was sheared into DNA fragments of about 200 bp using a Covaris ultrasonic crusher (see step 2.1). The library was prepared using the Illumina V3 VAHTS Universal DNA Library Preparation Kit (Vazyme ND607-02). The library was quantified using the dsDNA HS Assay Kit (Thermo Fisher) (see step 2.2). The library was captured using the xGen Hybridization and Capture Kit (Integrated DNA Technologies, 1080584) using FCS probes. After capture, library quality control (QC) was performed using a Qubit fluorometer (Invitrogen) and a LabChip GX Touch HT (Perkin Elmer). Finally, sequencing (2 × 150 bp) was performed on the NovaSeq6000 platform (Illumina) (see step 2.4).

[0106] The following is a detailed description of each experimental step:

[0107] 2.1 Sample preparation

[0108] Ten mutant gDNA samples containing known mutations in six genes (LPL, APOC2, LMF1, GPIHBP1, APOA5, and GPD1) were selected (see Appendix 5 for specific gene mutation types), as well as 10 wild-type gDNA samples that did not contain any mutations in the above six genes (Table 5). Approximately 300 ng of each gDNA sample was taken and the volume was made up to 50 μL with IDTE buffer (10 mM Tris, 0.1 mM EDTA). The gDNA samples were fragmented for 180 s using a Covaris ultrasonic disruptor set according to the parameters in Table 6 below.

[0109] Table 5. Sample information

[0110] Table 6. Covaris ultrasonic crusher parameter settings

[0111] 2.2 Library construction

[0112] 2.2.1 End-repair plus A

[0113] 1) Transfer the fragmented sample to a 0.2 mL sterilized centrifuge tube. Place the tube on ice and add the following reagents according to the table below.

[0114] Table 7

[0115] 2) After microcentrifugation, collect the liquid at the bottom of the tube and proceed with the following reaction.

[0116] Table 8

[0117] 2.2.2 Connector connection

[0118] 1) Thaw the ligase buffer, mix thoroughly by inversion, and place on ice until ready for use.

[0119] 2) Prepare the reaction system according to the table below and dispense the reagents into the above reaction tubes on ice.

[0120] Table 9

[0121] The IDT UDI Adapter Kit is recommended for adapters. If the cDNA input amount is ≤10 ng, dilute the adapter 20-fold (using Nuclease-Free Water) before use.

[0122] 3) After carefully confirming the sample ID, add 2 μL of Adapter to each sample, gently pipette to mix, and microcentrifuge to collect the liquid at the bottom of the tube. Perform the amplification reaction according to the following protocol.

[0123] Table 10

[0124] 2.2.3 Purification of ligation products

[0125] 1) Equilibrate the magnetic beads at room temperature for 30 minutes, vortex thoroughly to mix, and transfer 80 μL to a new 1.5 mL centrifuge tube.

[0126] 2) Transfer 100 μL of the ligation product to the 1.5 mL centrifuge tube prepared in step 2.2.2, vortex to mix, and incubate at room temperature for 5 minutes.

[0127] 3) Place the 1.5 mL centrifuge tube on a magnetic rack and let it stand until the solution is completely clear. Discard the supernatant.

[0128] 4) Add 200 μL of freshly prepared 80% (v / v) ethanol, incubate at room temperature for 30 seconds, and discard the supernatant.

[0129] 5) Repeat the previous step.

[0130] 6) Place the 1.5 mL centrifuge tube on a magnetic stand and let it sit for 1 minute. Discard any remaining solution and air dry at room temperature with the lid open until the ethanol is completely evaporated.

[0131] 7) Add 22 μL of Nuclease-Free Water, vortex to mix, incubate at room temperature for 2 min, microcentrifuge, and place on a magnetic rack. After the solution is completely clarified, transfer 20 μL of the supernatant to a new, labeled 0.2 mL centrifuge tube.

[0132] 2.2.4 Library Amplification

[0133] 1) Thaw the library construction amplification primers and library construction amplification buffer and mix thoroughly by inversion.

[0134] 2) Prepare the reaction system according to the table below and dispense into the above-mentioned connected purified product tubes.

[0135] Table 11

[0136] 3) Gently pipette to mix, then microcentrifuge to collect the liquid at the bottom of the tube. Amplify according to the following procedure:

[0137] Table 12

[0138] 2.2.5 PCR product purification

[0139] 1) Equilibrate the magnetic beads at room temperature for 30 minutes, vortex thoroughly to mix, and transfer 50 μL to a new 1.5 mL centrifuge tube.

[0140] 2) Transfer 50 μL of PCR product to the 1.5 mL centrifuge tube prepared in step 2.2.4, vortex to mix, and incubate at room temperature for 5 minutes.

[0141] 3) Place the 1.5 mL centrifuge tube on a magnetic rack and let it stand until the solution is completely clear. Discard the supernatant.

[0142] 4) Add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for 30 seconds, and discard the supernatant.

[0143] 5) Repeat the previous step.

[0144] 6) Microcentrifuge the 1.5 mL centrifuge tube from step 5) and place it on a magnetic rack. Let it stand for 1 minute, then discard the remaining solution. Open the lid and air dry at room temperature until the ethanol is completely evaporated.

[0145] 7) Add 32 μL of Nuclease-Free Water, vortex to mix, incubate at room temperature for 2 minutes, microcentrifuge, and place on a magnetic rack. After the solution is completely clarified, transfer 30 μL of the supernatant to a new labeled 1.5 mL centrifuge tube. This is the library product.

[0146] 2.2.6 Library Quality Control

[0147] 1) Using the Qubit nucleic acid quantification kit TM The dsDNA HS Assay Kit and its supporting instruments are used to determine the concentration of the DNA library. The total amount of DNA library should be ≥500 ng.

[0148] 2) Determine the DNA library fragment length using the DNA High Sensitivity Reagent Kit and its accompanying instrument. The main peak should be between 200 and 800 bp, with no obvious small or large fragments.

[0149] 2.3 Target region enrichment

[0150] 2.3.1 Library pooling and drying

[0151] 1) After carefully confirming the sample IDs as described in Table 3 or Table 4, combine the DNA libraries into a pool in a new 1.5 mL LoBind centrifuge tube.

[0152] 2) Prepare Blocker Master Mix in a new 1.5 mL LoBind centrifuge tube according to the table below. Add 10% if multiple samples are needed.

[0153] Table 13

[0154] 3) After mixing, add 6 μL Blocker Master Mix to each pool.

[0155] 4) Vortex thoroughly and centrifuge to collect the mix at the bottom of the tube. Seal the tube with sealing film, pierce 5 small holes in the film, and place in a vacuum concentrator to drain.

[0156] 2.3.2 Library Resolubilization, Denaturation, and Hybridization

[0157] 1) Prepare the Hybridization Master Mix in a new 1.5 mL LoBind centrifuge tube according to the table below. Prepare more for multiple samples.

[0158] Table 14

[0159] 2) After vortexing and centrifuging, add 17 μL of Hybridization Master Mix to the drained microcentrifuge tube (step 2.3.1). Vortex thoroughly and microcentrifuge to collect the solution at the bottom of the well. Let it stand at room temperature for 5-10 minutes to reconstitute. Transfer all 17 μL of the mix to a pre-prepared 0.2 μL microcentrifuge tube and place it in a PCR machine. After a brief centrifugation, place the tube back in the PCR machine and start the HYB program. Incubate the reaction at 65°C overnight for capture. The reaction system is as follows:

[0160] Table 15

[0161] 2.3.3 Preparation of washing buffers

[0162] 1) Prepare 1× working solution according to the table below. No additional preparation is required.

[0163] Table 16

[0164] 2) In a new 1.5 mL LoBind centrifuge tube, prepare Bead Resuspension Mix according to the table below.

[0165] Table 17

[0166] 2.3.4 Washing Streptavidin M270 Magnetic Beads

[0167] 1) Remove the magnetic beads at least 30 minutes in advance and equilibrate them to room temperature. Vortex mix thoroughly for about 15 seconds.

[0168] 2) According to the number of samples, take 50 μL / pool into a new 1.5 mL centrifuge tube.

[0169] 3) Add 100 μL / pool of mixed 1× Bead Wash Buffer to each centrifuge tube according to the CAP number. Gently pipette and mix 10 times, then place on a magnetic stand. After the solution is completely clarified, aspirate and discard the supernatant (magnetic beads adsorption for 1 min).

[0170] 4) Repeat the previous step 2 times.

[0171] 5) Add 17 μL / pool Bead Resuspension Mix to resuspend the magnetic beads, mix thoroughly, and collect the resuspension at the bottom of the well by microcentrifugation.

[0172] 6) Aliquot 17 μL of the magnetic bead mix into new 0.2 μL centrifuge tubes.

[0173] 2.3.5 Magnetic Bead Hybridization

[0174] 1) Heat 1× Wash Buffer 1 and 1× Stringent Wash Buffer in a 65°C metal bath at least 15 minutes in advance.

[0175] 2) After the hybridization reaction has proceeded overnight, remove the sample from the PCR instrument and microcentrifuge the sample.

[0176] 3) Close the HYB program and open the WASH program.

[0177] 4) Transfer all 17 μL of magnetic beads (preheated in advance) to the centrifuge tube for hybridization reaction, vortex to mix, centrifuge lightly, and then place in the PCR instrument.

[0178] 5) Incubate for 45 minutes. Vortex the tube every 12 minutes to mix thoroughly and avoid splashing the solution.

[0179] Table 18

[0180] 2.3.6 Heating and washing

[0181] 1) After the 45-minute incubation, remove the sample from the PCR instrument.

[0182] 2) Transfer 100 μL of preheated 1× Wash Buffer 1 to the sample and mix thoroughly by pipetting 10 times to avoid excessive bubbles.

[0183] 3) Transfer all the above mix to a new 1.5 mL centrifuge tube and place the sample on a magnetic stand for about 1 minute. After the solution is completely clarified, discard the supernatant.

[0184] 4) Remove the sample from the magnetic stand, transfer 150 μL of preheated 1× Stringent Wash Buffer to the sample, and mix by pipetting 10 times.

[0185] 5) After incubating in a metal bath at 65°C for 5 minutes, place the sample on a magnetic stand and discard the supernatant after the solution is completely clarified.

[0186] 6) Repeat steps 4) to 5).

[0187] 2.3.7 Washing at room temperature

[0188] 1) Add 150 μL of mixed 1× Wash Buffer 1, vortex to mix, and incubate for 2 minutes. During this period, let it stand for 30 seconds and shake it for 30 seconds. After a brief centrifugation, place it on a magnetic stand (about 1 minute). After the solution is completely clarified, aspirate and discard the supernatant.

[0189] 2) Add 150 μL of mixed 1× Wash Buffer 2, vortex to mix, and incubate for 2 minutes. During this period, let it stand for 30 seconds and shake it for 30 seconds. After a brief centrifugation, place it on a magnetic stand (about 1 minute). After the solution is completely clarified, aspirate and discard the supernatant.

[0190] 3) Add 150 μL of mixed 1× Wash Buffer 3, vortex to mix, and incubate for 2 minutes. During this period, let it stand for 30 seconds and shake it for 30 seconds. After a brief centrifugation, place it on a magnetic stand (about 1 minute). After the solution is completely clarified, aspirate and discard the supernatant.

[0191] 4) After re-centrifugation, use a 10 μL pipette tip to aspirate and discard all the residual 1× Wash Buffer 3.

[0192] 5) Remove the samples from the magnetic rack and add 20 μL of ultrapure water to each centrifuge tube. Mix thoroughly by pipetting 10 times to resuspend the magnetic beads. Carefully confirm the sample number and transfer all the resuspended solution to a new, labeled 0.2 mL centrifuge tube.

[0193] 2.3.8 PCR amplification

[0194] 1) Prepare the Amplification Reaction Mix in a new LoBind centrifuge tube. If multiple samples are needed, add 20% of the mixture.

[0195] Table 19

[0196] 2) Transfer 30 μL of the Amplification Reaction Mix (step 2.3.8) to a 0.2 mL centrifuge tube, vortex to mix, microcentrifuge, and place in a PCR instrument. Perform the reaction according to the following protocol.

[0197] Table 20

[0198] 2.3.9 Post-PCR Purification

[0199] 1) Prepare 400 μL of fresh 80% ethanol for each sample. If multiple samples are needed, add 10% ethanol.

[0200] 2) Aliquot the AMpure XP Beads that have been equilibrated to room temperature into new 1.5 mL centrifuge tubes, 75 μL each.

[0201] 3) After carefully verifying the number, transfer all amplified products to a 1.5 mL centrifuge tube containing magnetic beads. Vortex and microcentrifuge. Incubate at room temperature for 5-10 minutes, then place on a magnetic rack. Once the solution is completely clear, aspirate and discard the supernatant.

[0202] 4) Add 200 μL of freshly prepared 80% ethanol, let it stand for 1 min, and then discard the supernatant.

[0203] 5) Repeat step 4).

[0204] 6) Leave the sample on the magnetic rack and let the beads dry for 1-3 minutes.

[0205] 7) Add 27 μL of ultrapure water to the centrifuge tube, vortex thoroughly to mix, and microcentrifuge. Incubate at room temperature for 5 minutes, and place the centrifuge tube on a magnetic rack.

[0206] 8) After carefully confirming the sample ID, transfer 25 μL of supernatant to a newly labeled 1.5 mL centrifuge tube.

[0207] 2.4 Sequencing

[0208] The sequencing experiments of the above 10 samples were completed according to the operating instructions of the NovaSeq6000 (Illumina) sequencer and the NovaSeq 6000v2 kit sequencing reagent kit.

[0209] The sequencing data were analyzed by bioinformatics methods, and the test results are shown in Appendix 21 and Table 22:

[0210] Table 21. Detection results of 10 mutant gDNA samples containing 6 known gene mutation types

[0211] Table 22. Test results for 10 wild-type gDNA samples that do not contain any variant types of the 6 genes

[0212] From the above data, it can be seen that the method of this embodiment has a sensitivity of 100% and a specificity of 100% for detecting mutations in the six genes characteristic of FCS (LPL, APOC2, LMF1, GPIHBP1, APOA5 and GPD1), and has extremely high detection accuracy.

[0213] Overall, the embodiments of the present application designed target capture probes covering all exons and hotspot intron regions of six FCS-related genes (LPL, APOC2, LMF1, GPIHBP1, APOA5 and GPD1) through a capture sequencing method. By detecting the six gene variation types closely related to FCS, accurate detection of FCS is achieved, which is of great significance for the clinical diagnosis and treatment of FCS.

[0214] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0215] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. In addition, it should be understood that after reading the above-mentioned teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the contents of the attached claims, and the description can be used to interpret the contents of the claims.

Claims

1. Capture probes, comprising one or more of probe combinations 1 to 6; Probe combination 1 includes one or more of probes 1 to 26; Probe combination 2 includes one or more of probes 27 to 64; Probe combination 3 includes one or more of probes 65 to 112; Probe combination 4 includes one or more of probes 113 to 139; Probe combination 5 includes one or more of probes 140 to 382; Probe combination 6 includes one or more of probes 383 to 425; The probes 1 to 425 are probes constructed for the target gene fragments between different start sites and end sites on the following chromosomes and are complementary to the target gene fragments: The reference genome is Hg19.

2. The capture probe according to claim 1, comprising multiple groups of probe combination 1 to probe combination 6; The probe combination 1 includes multiple probes from probe 1 to probe 26; The probe combination 2 includes multiple probes from probes 27 to probes 64; The probe combination 3 includes multiple probes from probe 65 to probe 112; The probe combination 4 includes multiple probes from the probes 113 to 139; The probe combination 5 includes multiple probes from the probes 140 to 382; The probe combination 6 includes multiple probes from probes 383 to probes 425 .

3. The capture probe according to claim 2, comprising probe combination 1 to probe combination 6; The probe combination 1 includes the probes 1 to 26; The probe combination 2 includes the probes 27 to 64; The probe combination 3 includes the probes 65 to 112; The probe combination 4 includes the probes 113 to 139; The probe combination 5 includes the probes 140 to 382; The probe combination 6 includes the probes 383 to 425 .

4. Use of the capture probe according to any one of claims 1 to 3 in the preparation of a familial chylomicronemia syndrome detection kit.

5. A familial chylomicronemia syndrome detection kit comprising the capture probe according to any one of claims 1 to 3.

6. The familial chylomicronemia syndrome detection kit according to claim 5, further comprising one or more of nucleic acid extraction reagents, DNA library construction reagents, sample and library quantification reagents, fragment quality control reagents, hybridization capture reagents, nucleic acid purification reagents, target gene fragment amplification reagents and sequencing reagents.

7. Use of the capture probe according to any one of claims 1 to 3 in constructing a library for detecting gene mutations associated with familial chylomicronemia syndrome.

8. Use of the detection kit according to claim 5 or 6 in constructing a familial chylomicronemia syndrome-related gene mutation detection library.

9. A method for detecting a familial chylomicronemia syndrome-related gene mutation, comprising the steps of using the capture probe described in any one of claims 1 to 3 or the detection kit described in claim 5 or 6 to capture a target gene fragment containing a familial chylomicronemia syndrome-related gene mutation, and detecting the captured fragment.

10. The method for detecting gene mutations associated with familial chylomicronemia syndrome according to claim 9, wherein: The captured fragments are detected by sequencing.