Method for constructing a universal gene detection library of familial hypercholesterolemia and kit

Abstract

The application belongs to the technical field of gene mutation detection, and discloses a construction method and a kit of a universal gene detection library of familial hypercholesterolemia. The construction method and the kit are based on a multiplex PCR amplicon sequencing technology, involve four single gene mutations of LDLR, APOB, PCSK9 and LDLRAP1, and 12 gene sites related to familial hypercholesterolemia, i.e. rs2479409, rs629301, rs1367117, rs4299376, rs1564348, rs1800562, rs3787354, rs11220462, rs8017377, rs6511720, rs429386 and rs7412. The method and the kit can be compatible with multiple sequencing platforms, have the advantages of simple library construction steps, cost saving and wide application range, and the like.

Description

Technical Field

[0001] This invention belongs to the field of gene mutation detection technology, specifically relating to a method for constructing a universal gene detection library for familial hypercholesterolemia and its reagent kit. Background Technology

[0002] Familial hypercholesterolemia (FH) is a lipid metabolism disorder, the main clinical manifestation of which is a significant increase in serum low-density lipoprotein cholesterol (LDL-C) levels, skin / tendon xanthelasma, corneal rings, etc. Because FH patients are exposed to high serum LDL-C levels from birth, they have a significantly increased risk of atherosclerotic cardiovascular disease (ASCVD).

[0003] Familial hypercholesterolemia (FH) has an incidence rate of 1 / 500 in the general population, and its prevalence is as high as 23.6% in individuals with early-onset myocardial infarction. Most cases of FH are caused by genetic factors, including single-gene mutations and polygenic mutations. Single-gene FH is caused by four genes: LDLR, APOB, PCSK9, and LDLRAP1. Single-gene FH is primarily dominant, with recessive inheritance being rare. It includes two types: heterozygous FH (HeFH) and homozygous FH (HoFH), with heterozygous FH being more common. In patients clinically diagnosed with FH, only about 40% have detectable pathogenic or potentially pathogenic variants in these four genes. For the remaining 80% of clinically diagnosed patients who test negative for these four genes, a polygenic model can be used to explain the condition. This model uses 12 single nucleotide polymorphisms (SNPs) and employs a scoring method for interpretation. Polygenic FH does not follow Mendelian inheritance. For patients with familial hypercholesterolemia, only diseases caused by a single gene require familial cascade screening.

[0004] Currently, the most commonly used methods for detecting familial hypercholesterolemia are whole-exome sequencing and liquid-phase capture sequencing. Both of these methods combine probe capture library preparation with next-generation sequencing. However, these methods have the following drawbacks: cumbersome experimental procedures, long detection cycles, high costs, and the presence of GC-enriched regions, making them difficult to meet clinical testing requirements.

[0005] Currently, there are also publicly available patents that use PCR methods to detect gene mutations in familial hypercholesterolemia, such as the patent "A method for constructing a universal gene detection library for hereditary familial hypercholesterolemia and its kit". However, this patent only detects single-gene familial hypercholesterolemia and cannot explain the cause of disease in patients with four single-gene recessive mutations. Secondly, its library construction and sequencing analysis has a low Q30 percentage, which means that its coverage and uniformity of the above-mentioned genes are poor, and there is a possibility of missed detection.

[0006] Therefore, there is a need in the art for methods and kits that can be used to simultaneously detect monogenic and polygenic familial hypercholesterolemia, and that are highly sensitive, high-throughput, and low-cost. Summary of the Invention

[0007] In order to solve the above-mentioned problems in the existing technology, the present invention aims to provide a method for constructing a universal gene detection library for familial hypercholesterolemia and a kit thereof.

[0008] The technical solution adopted in this invention is: a method for constructing a universal gene detection library for familial hypercholesterolemia, comprising the following steps:

[0009] S1. First round of multiplex PCR reaction: including T1 reaction tube system, T2 reaction tube system and T3 reaction tube system; the T1 reaction tube system includes a first primer pool and a first reaction solution; the T2 reaction tube system includes a second primer pool and a second reaction solution; the T3 reaction tube system includes a third primer pool and a third reaction solution;

[0010] The first primer pool includes 86 pairs of primers, and the nucleotide sequences of the upstream and downstream primers of the 86 pairs of primers are shown in SEQ ID NO.1-SEQ ID NO.172, respectively.

[0011] The second primer pool includes 87 pairs of primers, and the nucleotide sequences of the upstream and downstream primers of the 87 pairs of primers are shown in SEQ ID NO.173-SEQ ID NO.346, respectively.

[0012] The third primer pool includes 15 pairs of primers, and the nucleotide sequences of the upstream and downstream primers of the 15 pairs of primers are shown in SEQ ID NO.347-SEQ ID NO.377, respectively.

[0013] S2. Combining multiplex PCR reactions: Run the PCR amplification reaction in the T1 reaction tube system to obtain reaction product A; run the PCR amplification reaction in the T2 reaction tube system to obtain reaction product B; run the PCR amplification reaction in the T3 reaction tube system to obtain reaction product C; mix and combine the above reaction products A, B and C to obtain the combined product.

[0014] S3. Purification of the combined products using magnetic beads;

[0015] S4, second round of sequence PCR reaction, second round of magnetic bead purification and library concentration measurement and quality control, to complete library construction.

[0016] Preferably, the volume of the T1 reaction tube system is 10-12.5 μL;

[0017] The volume of the T2 reaction tube system is 10-12.5 μL;

[0018] The volume of the T3 reaction tube system is 1-5 μL.

[0019] Preferably, the volume of the second round of sequence PCR reaction system is 30-40 μL.

[0020] Preferably, in the multiplex PCR reaction merging, the volumes of reaction product A, reaction product B, and reaction product C during the preparation of the merged product are 11.5 μL, 13.5 μL, and 5 μL, respectively.

[0021] Preferably, the first reaction solution includes Enhancer buffer NB buffer, Enhancer buffer M buffer, IGT-EM808 enzyme mixture, gDNA and ddH2O;

[0022] The second reaction solution includes Enhancer buffer NB buffer, Enhancer buffer M buffer, IGT-EM808 enzyme mixture, gDNA, and ddH2O.

[0023] Preferably, the third reaction solution includes IGT-GC Buffer, IGT-GC Enhancer Buffer, IGT-GC dNTP, Enhancer Buffer (NB), IGT-GC Polymerase, gDNA, and ddH2O.

[0024] Preferably, the magnetic bead purification process in the purified and combined products is as follows:

[0025] Add magnetic beads to the merged products, mix well, incubate at room temperature, remove the supernatant, add YF buffer B and mix well, incubate at room temperature a second time, remove the supernatant, add ethanol solution, let stand, add Nuclease-free water, resuspend the magnetic beads, let stand at room temperature, aspirate the supernatant and transfer it to a new PCR tube. The supernatant in the tube is the merged multiplex PCR product.

[0026] Preferably, the second round of magnetic bead purification process is as follows: add magnetic beads to the PCR reaction system, mix well, incubate at room temperature, remove the supernatant, add YF buffer B, mix well, incubate at room temperature a second time, let stand, add Nuclease-free water or 1×TE buffer (pH 8.0), mix well, resuspend the magnetic beads, aspirate the supernatant, and transfer it to a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library.

[0027] A universal genetic testing kit for familial hypercholesterolemia, comprising the T1 reaction tube system, T2 reaction tube system and T3 reaction tube system constructed as described above.

[0028] The beneficial effects of this invention are as follows:

[0029] (I) This invention employs multiplex PCR amplicon library construction technology to simultaneously capture and construct libraries from the whole exon regions of the four pathogenic genes (LDLR, APOB, PCSK9, LDLRAP1) of monogenic familial hypercholesterolemia, as well as 20 bp upstream and downstream of these genes and 12 multi-gene pathogenic loci (rs2479409; rs629301; rs1367117; rs4299376; rs1564348; rs1800562; rs3787354; rs11220462; rs8017377; rs6511720; rs429386; rs7412).

[0030] (II) This invention patent employs MultipSeq multiplex amplicon capture technology, using a two-step PCR amplification method to complete the amplification of the target region and library construction. This technology can rapidly target and linearly amplify thousands or even tens of thousands of mutation sites such as SNPs and Indels while ensuring amplification uniformity, followed by sequencing and deep analysis using mainstream sequencing platforms.

[0031] (III) The kit uses two rounds of PCR reaction for library construction, which has the advantages of short library construction cycle, high comparison rate, good uniformity, good reproducibility and simple operation. In addition, the invention can also use a variety of samples for library construction, such as saliva gDNA, blood gDNA, etc.

[0032] (iv) The above-mentioned method for constructing the detection library simplifies the entire process, reduces labor costs, reduces the amount of detection data, and reduces costs. Attached image description:

[0033] Figure 1 This diagram illustrates the measurement of fragment length and purity of library samples using the Qsep400 fully automated nucleic acid and protein analysis system during library quality testing. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. Those skilled in the art will understand that the embodiments described below are only some, not all, embodiments of the present invention, and are used merely to illustrate the invention, and should not be considered as limiting the scope of the invention. All reagents used are commercially available conventional products.

[0035] Example:

[0036] This invention patent employs MμLtipSeq multiplex amplicon capture technology, using a two-step PCR amplification method to complete target region amplification and library construction. This technology can rapidly target and linearly amplify thousands or even tens of thousands of mutation sites, such as SNPs and Indels, while ensuring amplification uniformity, followed by sequencing and deep analysis using mainstream sequencing platforms.

[0037] This invention contains multiple amplicones and consists of three reaction tubes: T1, T2, and T3 (three tubes in total). The kit uses two rounds of PCR reactions for library construction, which has advantages such as short library construction cycle, high alignment rate, good uniformity, good reproducibility, and simple operation. In addition, this invention can also use various samples for library construction, such as saliva gDNA and blood gDNA.

[0038] The library construction process of this kit consists of several major steps: the first round of multiplex PCR reaction, product merging, magnetic bead purification of the merged products, the second round of adapter sequence PCR reaction, magnetic bead purification, concentration measurement and quality control.

[0039] I. First round of multiplex PCR reaction

[0040] (1) Multiplex PCR reaction system

[0041] The first round of multiplex PCR was performed in three reaction tubes, using primer pool T1, primer pool T2, and primer pool T3. All other reagents in the three primer pool tubes were the same.

[0042] T1 and T2 reaction tube systems (30 μL):

[0043]

[0044] Note: The initial amount of gDNA is 50 ng / tube; the DNA concentration is based on the quantification results from Qubit (Life Technologies).

[0045] (2) Multiplex PCR reaction conditions

[0046] T1 / T2 PCR instrument program: Hot lid 105℃

[0047] 95℃ for 3 min 30 s, 98℃ for 20 s, cycle 18 times; 60℃ for 4 min, 72℃ for 5 min.

[0048] T3 PCR instrument program: Hot lid 105℃

[0049] 95℃ for 3 min 30 s, 98℃ for 20 s, repeat 20 times; 60℃ for 2 min, 72℃ for 5 min.

[0050] II. Combining of products from the first round of PCR reaction

[0051] After the first round of PCR reaction, the PCR products from the three reaction tubes were combined according to the table below, with a total volume of 30 μL.

[0052]

[0053]

[0054] III. Purification and Combination of Products Using Magnetic Beads

[0055] (1) Add 27 μL of AMPure XP magnetic beads that have been equilibrated at room temperature to 30 μL of PCR pooled product and gently pipette and mix 20 times.

[0056] (2) After incubating at room temperature for 5 min, place the PCR tube on the DynaMag-96Side magnetic rack for 3 min;

[0057] (3) Thoroughly remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B into the tube and gently pipette and mix 20 times.

[0058] (4) After incubating at room temperature for 5 min, place the PCR tube on the DynaMag-96Side magnetic rack for 3 min;

[0059] (5) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μL of 80% ethanol solution to the tube, and let stand for 30 seconds;

[0060] (6) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μL of 80% ethanol solution to the tube, let it stand for 30 seconds and then completely remove the supernatant (it is recommended to use a 10 μL pipette to remove the residual ethanol solution at the bottom).

[0061] (7) Let stand at room temperature for 3 minutes to allow the residual ethanol to evaporate completely;

[0062] (8) Remove the PCR tube from the magnetic rack, add 24 μL of Nuclease-free water, gently pipette to resuspend the magnetic beads to avoid generating air bubbles, and let stand at room temperature for 2 min.

[0063] (9) Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0064] (10) Use a pipette to draw 13.5 μL of supernatant and transfer it to a new 200 μL PCR tube. The supernatant in the tube is the combined multiplex PCR product.

[0065] IV. Second Round of PCR Reaction with Adapter Sequences

[0066] (1) Reaction system

[0067] Reagent (components) Volume (μL) PCR product mixture 13.5 Enhancer buffer M 2.5 <![CDATA[ddH2O]]> 2 IGT-I5 Index (10µM) 1 IGT-I7 Index (10µM) 1 IGT-EM808 polymerase mixture 10

[0068] Note: The PCR product mixture is the multiplex PCR product purified in the previous step.

[0069] (2) Reaction conditions

[0070] Run the PCR instrument program: Heat lid 105℃

[0071] 95℃ for 3 min 30 s, 98℃ for 20 s, repeat 9 times; 60℃ for 1 min, 72℃ for 5 min.

[0072] V. Second round of magnetic bead purification

[0073] (1) Add 27 μL of AMPure XP magnetic beads that have been equilibrated at room temperature to a 30 μL PCR reaction system and gently pipette and mix 20 times.

[0074] (2) After incubating at room temperature for 5 min, place the PCR tube on the DynaMag-96Side magnetic rack for 3 min;

[0075] (3) Thoroughly remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, and gently pipette and mix 20 times.

[0076] (4) After incubating at room temperature for 5 min, place the PCR tube on the DynaMag-96Side magnetic rack for 3 min;

[0077] (5) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μL of 80% ethanol solution to the tube, and let stand for 30 seconds;

[0078] (6) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μL of 80% ethanol solution to the tube, let stand for 30 seconds and then completely remove the supernatant.

[0079] (7) Let stand at room temperature for 3 minutes to allow the residual ethanol to evaporate completely;

[0080] (8) Remove the centrifuge tube from the magnetic rack, add 24 μL of Nuclease-free water or 1×TE buffer (pH 8.0), gently pipette and mix 20 times, resuspend the magnetic beads to avoid generating air bubbles, and let stand at room temperature for 2 minutes.

[0081] (9) Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0082] (10) Use a pipette to draw 20 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library.

[0083] VI. Quantitative Library

[0084] Take 2 μl of the library and use the Qubit 3.0 Fluorometer (Qubit dsDNA HS Assay Kit) to determine the library concentration and record the concentration.

[0085] VII. Document Quality Inspection

[0086] like Figure 1 As shown, 1 μl of library sample was taken and the Qsep400 fully automated nucleic acid and protein analysis system was used to measure the library fragment length and purity. The horizontal axis represents time, and the vertical axis represents the relative fluorescence unit, i.e., the acquired signal intensity.

[0087] T1 primer pool primer sequence list:

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] T2 primer pool primer sequence list:

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] T3 primer pool primer sequence list:

[0103]

[0104]

[0105] Experimental Example

[0106] 1. Test Sample

[0107] The sample tested was taken from a volunteer's blood.

[0108] 2. Detection Method

[0109] DNA extraction: DNA is extracted from the sample to be tested using standard methods as a template.

[0110] PCR amplification:

[0111] The amplification reaction system for the first round is the same as the amplification system for the first round of PCR reaction described above.

[0112] The amplification conditions for the first round of PCR reaction are the same as those described above.

[0113] The purification steps for the product after the first round of PCR amplification are the same as those described above for the purification of the product after the first round of PCR amplification.

[0114] After the first round of PCR reaction, the PCR products from the three tubes were combined as described above.

[0115] The second round of PCR amplification system is the same as the second round of PCR reaction amplification system described above.

[0116] The amplification conditions for the second round of PCR reaction are the same as those described above.

[0117] The purification steps for the products after the second round of PCR amplification are the same as those for the products after the first round of PCR amplification described above.

[0118] The library after the second round of purification was quantified as described in the library quantification steps above.

[0119] The library after the second round of purification underwent quality testing, as described above. The target fragment distribution ranged from 280bp to 420bp.

[0120] 3. Test Results

[0121] The following are the actual sequencing data of the qualified libraries constructed using this kit on the Illumina Nova 6000 (NEXTSeq550) sequencing platform. The libraries were then sequenced, and bioinformatics analysis was performed. First, the sequencing data quality was assessed. The amount of sequencing data after assessment was statistically analyzed, and reference sequence alignment and depth data were performed. The final results are shown in the table below:

[0122]

[0123] Note: Raw base (Mb): Size of the raw sequencing data

[0124] QCrate (%): Quality Control Rate

[0125] Total reads mapping rate (%): Total read length matching efficiency

[0126] Total read capture rate (%): Total read capture efficiency

[0127] Coverage rate (%): Coverage

[0128] Target mean depth: average sequencing depth of the target region

[0129] T 30X coverage rate (%): Coverage at sequencing depths of 30X or higher.

[0130] T 20X coverage rate (%): Coverage at sequencing depths of 30X or higher.

[0131] The above method was used to sequence two samples that were positive for whole-exome sequencing, verifying the accuracy of the method. The sample detection results are shown in the table below:

[0132]

[0133] This invention is not limited to the optional embodiments described above. Anyone inspired by this invention can derive various other products, all of which fall within the scope of protection of this invention. The specific embodiments described above should not be construed as limiting the scope of protection of this invention. Those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features, without departing from the scope of this invention. At the same time, these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this invention.

Claims

1. A method for constructing a universal gene detection library for familial hypercholesterolemia, characterized in that, Includes the following steps: S1. First round of multiplex PCR reaction: including T1 reaction tube system, T2 reaction tube system and T3 reaction tube system; the T1 reaction tube system includes a first primer pool and a first reaction solution; the T2 reaction tube system includes a second primer pool and a second reaction solution; the T3 reaction tube system includes a third primer pool and a third reaction solution; The nucleotide sequences of the upstream and downstream primers of the first primer pool are shown in SEQ ID NO.1-SEQ ID NO.116, respectively; The nucleotide sequences of the upstream and downstream primers of the second primer pool are shown in SEQ ID NO.117-SEQ ID NO.230, respectively; The nucleotide sequences of the upstream and downstream primers of the third primer pool are shown in SEQ ID NO.231-SEQ ID NO.236, respectively. S2. Multiplex PCR reaction merging: PCR amplification reaction was run in the T1 reaction tube system to obtain reaction product A; PCR amplification reaction was run in the T2 reaction tube system to obtain reaction product B. PCR amplification was performed using a T3 reaction tube system to obtain reaction product C; reaction products A, B and C were then mixed and combined to obtain the combined product. In the multiplex PCR reaction merging, the volumes of reaction product A, reaction product B, and reaction product C during the preparation of the merged product are 12.5 μL, 12.5 μL, and 5 μL, respectively. S3. Purification of the combined products using magnetic beads; S4, second round of sequence PCR reaction, second round of magnetic bead purification and library concentration measurement and quality control, to complete library construction; The volume of the second round of sequence PCR reaction system is 30-40 μL; The first reaction solution includes Enhancer buffer NB buffer, Enhancer buffer M buffer, IGT-EM808 enzyme mixture, gDNA, and ddH2O; The second reaction solution includes Enhancer buffer NB buffer, Enhancer buffer M buffer, IGT-EM808 enzyme mixture, gDNA, and ddH2O; The third reaction solution includes IGT-GC Buffer, IGT-GC Enhancer Buffer, IGT-GC dNTP, Enhancer Buffer (NB), IGT-GC Polymerase, gDNA, and ddH2O; The second round of magnetic bead purification process is as follows: add magnetic beads to the PCR reaction system, mix well, incubate at room temperature, remove the supernatant, add YF buffer B, mix well, incubate at room temperature a second time, let stand, add Nuclease-free water or 1×TE buffer at pH 8.0, mix well, resuspend the magnetic beads, aspirate the supernatant, and transfer it to a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library.

2. The method for constructing a universal gene detection library for familial hypercholesterolemia according to claim 1, characterized in that, The magnetic bead purification process for the purified and combined products is as follows: Add magnetic beads to the merged products, mix well, incubate at room temperature, remove the supernatant, add YF buffer B and mix well, incubate at room temperature a second time, remove the supernatant, add ethanol solution, let stand, add Nuclease-free water, resuspend the magnetic beads, let stand at room temperature, aspirate the supernatant and transfer it to a new PCR tube. The supernatant in the tube is the merged multiplex PCR product.

3. A general genetic test kit for familial hypercholesterolemia, characterized by, It includes the T1 reaction tube system, T2 reaction tube system and T3 reaction tube system in the construction method of any one of claims 1-2 above.