Primer sets, kits and systems for rapid diagnosis of PWS / AS

Abstract

The present invention belongs to the field of medical detection, and discloses a primer set, a kit and a system for rapid diagnosis of PWS / AS. The present invention uses MS-PCR in combination with NGS sequencing technology to detect the methylation level of the SNRPN gene in the 15q11.2-q13 region of chromosome 15. The detection method can be used for rapid molecular diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) epigenetic inheritance. The method is easy to operate, has low detection cost, good detection result accuracy, high detection sensitivity and specificity, and high detection throughput, which is suitable for clinical promotion and application. The detection results of this method can accurately distinguish the abnormal methylation level of the chromosome region 15q11.2-q13, can be used for rapid auxiliary diagnosis of PWS syndrome and AS syndrome, and is suitable for clinical rapid diagnosis applications.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection, and in particular relates to a primer set, a kit and a system for rapid diagnosis of Prader-Willi syndrome (PWS) / Angelman syndrome (AS). Background Art

[0002] Prader-Willi syndrome (PWS), also known as Prader-Willi syndrome, is a rare genetic disease with the main symptoms of hypotonia, intellectual disability, delayed gonadal development and obesity. Angelman syndrome (AS), also known as Angelman syndrome, is a neurogenetic disease caused by the absence or downregulation of maternal imprinted genes. Its main features include severe intellectual disability, language development disorder, movement or balance disorder, special facial features, etc.

[0003] Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are both caused by functional defects of imprinted genes and are related to genetic abnormalities on chromosome 15. Deletion of the q11-q13 region of chromosome 15, balanced translocation, or related gene mutations in this region may cause the disease. In the population, the incidence of PWS is 1 / 30,000-1 / 10,000, and the incidence of AS is about 1 / 50,000-1 / 24,000, and most cases are sporadic.

[0004] Genomic imprinting is an epigenetic phenomenon, which refers to the phenomenon that changes in a chromosome or the same gene can cause different phenotypes when passed from parents of different sexes to offspring. The most likely mechanism of this phenomenon is DNA methylation. PWS and AS have a common genetic background, but there are certain differences between the two in terms of genetic mechanism. PWS is usually caused by the deletion of a specific region on chromosome 15 inherited by an individual from the father. Other conditions include maternal uniparental disomy (no paternal chromosome 15) (UPD) and gene imprinting defect (ID). AS is usually caused by the deletion of a specific region on chromosome 15 inherited by an individual from the mother. Other conditions include paternal uniparental disomy (no maternal chromosome 15) (UPD), UBE3A gene mutation and gene imprinting defect (ID).

[0005] Technical defects and shortcomings of existing diagnostic methods:

[0006] At present, PWS and AS are tested for genes through high-resolution chromosome analysis (HRB), fluorescence in situ hybridization (FISH), methylation-specific polymerase chain reaction (MS-PCR), methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), chromosome microarray analysis (CMA), etc., and chromosome abnormalities or gene mutations can be found, which can be used as the basis for diagnosis. Among them, high-resolution chromosome analysis (HRB) can detect deletions in the chromosome region 15q11-q13, but cannot detect uniparental disomy (UPD), imprinted gene mutations and microdeletions, and the overall detection rate is low. Fluorescence in situ hybridization (FISH) is used to detect deletions in specific regions on chromosome 15, which can distinguish the genetic deletion causes of PWS and AS, but it also cannot detect UPD and imprinted gene mutations. In addition, the detection and operation of this technology are difficult and require high instrument, environment, and personnel operation. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) detects gene deletions or duplications, and has a high detection rate, but the development difficulty and cost of this technology are high. The chromosome microarray analysis (CMA) method can detect copy number variations of chromosomal imbalance, but it cannot distinguish whether the deletion comes from the father or the mother, and the overall detection rate is low. The methylation-specific polymerase chain reaction (MS-PCR) can detect deletions, UPD and imprinting defects in the chromosome region 15q11-q13 through DNA methylation analysis. However, the MS-PCR experimental method only detects the methylation level of a single site. For example, the q11.2-q13 region of chromosome 15 in the human body contains a total of 113 possible methylation sites, and the number of sites detected by MS-PCR is less than 1%, which may result in false negatives or false positives. Summary of the invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a primer set, a kit and a system for rapid diagnosis of PWS / AS.

[0008] The present invention provides a molecular biological detection method for rapid diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). The method can accurately detect abnormal methylation levels of chromosome region 15q11.2-q13 by detecting the methylation level of the SNRPN gene in the q11.2-q13 region of human chromosome 15. The method enriches the target gene by MS-PCR, and is easy to operate and inexpensive; combined with high-throughput sequencing technology, the detection result has high sensitivity, good accuracy and high throughput, and is suitable for clinical rapid diagnosis applications.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention designs 10 specific primers according to the q11.2-q13 region of chromosome 15 of the human genome, which are respectively used to amplify the genes SNRPN-CGI1, SNRPN-CGI2, SNRPN-CGI3, KLF4, and RHOG, wherein SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI3 are used as methylation level assessments of 15q11.2-q13, KLF4 is used as a quality control gene for the non-methylated region, and the RHOG gene is used as a quality control gene for the methylated region. After the whole genome DNA is methylated, MS-PCR amplification of 5 gene fragments is performed respectively, and after the ends of the 5 PCR products are repaired, an "A" base is added to the 3' end, and the linker is connected, and the library is amplified, and an NGS library is constructed and NGS sequencing is performed to analyze the methylation level of the 15q11.2-q13 region, thereby realizing rapid molecular diagnosis of PWS and AS according to the epigenetic methylation level.

[0011] In a first aspect, the present invention provides a primer set for rapid diagnosis of PWS / AS, wherein the primer set comprises the following primers:

[0012] Two specific primers for amplifying the SNRPN-CGI1 gene include:

[0013] Primer 1: 5′-gAgggAgTTgggATTTTTgTA-3′ (SEQ ID No. 1);

[0014] Primer 2: 5′-AAATCCTAAAAACTTAAAATATCTAATAAA-3′ (SEQ ID No. 2);

[0015] Two specific primers for amplifying the SNRPN-CGI2 gene include:

[0016] Primer 1: 5′-AATTAgATTYggAATgTTTAgAgg-3′ (SEQ ID No. 3);

[0017] Primer 2: 5′-CAATAATCACTATTATACACCTACCTA-3′ (SEQ ID No. 4).

[0018] Two specific primers for amplifying the SNRPN-CGI3 gene include:

[0019] Primer 1: 5′-TTAAAgTgTTgTTTgggAAAggA-3′ (SEQ ID No. 5).

[0020] Primer 2: 5′-AATACCTAACCTCTAACCACATTCC-3′ (SEQ ID No. 6).

[0021] The second aspect of the present invention provides use of the primer set described in the first aspect in preparing a kit for rapid diagnosis of PWS / AS.

[0022] In one embodiment, the method for rapid diagnosis of PWS / AS comprises the following steps:

[0023] (1) Provide genomic DNA to be tested;

[0024] (2) performing methylation conversion on the genomic DNA to be detected in step (1) to obtain converted genomic DNA;

[0025] (3) using the transformed genomic DNA of step (2) as a template, performing multiplex MS-PCR amplification using the primer set according to the first aspect, the non-methylated region quality control gene primer set, and the methylated region quality control gene primer set;

[0026] (4) Construction of NGS library;

[0027] (5) Perform NGS library sequencing and analyze the average methylation ratios of SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI13 in the 15q11.2-q13 region, and interpret the results.

[0028] In one embodiment, the multiplex MS-PCR amplification products can be further purified before the next step of reaction; the purification is performed using DNA purification magnetic beads.

[0029] In one embodiment, the non-methylated region quality control gene is the KLF4 gene, and the methylated region quality control gene is the RHOG gene;

[0030] Two specific primers for amplifying the KLF4 gene include:

[0031] Primer 1: 5′-gAgAATAAAgTTTAggTTTAggAgAT-3′ (SEQ ID No. 7).

[0032] Primer 2: 5′-ATAACAACAAAAAACCACCACTAAC-3′ (SEQ ID No. 8).

[0033] Two specific primers for amplifying the RHOG gene include:

[0034] Primer 1: 5′-TATATTTTTggATgTTATTTgTgT-3′ (SEQ ID No. 9).

[0035] Primer 2: 5′-TAAACCTAAACCTATAAAACACTAC-3′ (SEQ ID No. 10).

[0036] The average methylation ratio of the KLF4 gene region amplified by SEQ ID No. 7 and 8 was used as the non-methylation internal reference; the average methylation ratio of the RHOG gene region amplified by SEQ ID No. 9 and 10 was used as the methylation internal reference.

[0037] In a third aspect, the present invention provides a kit for rapid diagnosis of PWS / AS, comprising the primer set described in the first aspect above;

[0038] The kit also includes the non-methylated region quality control gene primer set, the methylated region quality control gene primer set, DNA polymerase, reaction buffer, etc. described in the second aspect.

[0039] In a fourth aspect, the present invention provides a device for rapid diagnosis of PWS / AS, comprising: the kit described in the third aspect; and a kit for constructing an NGS library.

[0040] In one embodiment, the kit for constructing an NGS library includes an end repair enzyme, a linker, a ligase, DNA purification magnetic beads, a library PCR primer, and a library PCR reaction buffer.

[0041] In a fifth aspect, the present invention provides a method for rapid diagnosis of PWS / AS, comprising the following steps:

[0042] 1) Provide genomic DNA to be tested;

[0043] 2) performing methylation conversion on the genomic DNA to be detected in step 1) to obtain converted genomic DNA;

[0044] 3) using the transformed genomic DNA from step 2) as a template, performing multiplex MS-PCR amplification using the primer set according to the first aspect and the primer set for quality control genes in the non-methylated region and the primer set for quality control genes in the methylated region described in the second aspect;

[0045] 4) Construction of NGS library;

[0046] 5) Perform NGS library sequencing and analyze the average methylation ratios of SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI13 in the 15q11.2-q13 region, and interpret the results.

[0047] In one embodiment, the genomic DNA is genomic DNA from a whole blood sample, and preferably a commercial DNA extraction kit can be used.

[0048] In one embodiment, the methylation conversion is a methylation enzymatic conversion; preferably, a commercial enzyme conversion kit can be used.

[0049] In one embodiment, the multiplex MS-PCR amplification products can be further purified before the next step of reaction; the purification is performed using DNA purification magnetic beads.

[0050] In one embodiment, the construction of the NGS library can be carried out using a commercial NGS library preparation kit.

[0051] In one embodiment, the NGS library sequencing is performed using a second-generation high-throughput sequencer; different sequencing platforms can be adapted according to the type of NGS library, and the amount of sequencing data on each sample library is 1.0G. Further selected from BGI DNBSEQ-T7 high-throughput sequencing.

[0052] In one embodiment, the results are interpreted and the results are comprehensively determined based on the average methylation ratios of SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI13 in the 15q11.2-q13 region:

[0053] Interpretation criteria:

[0054] Negative samples: methylation level is 50% ± 20%.

[0055] PWS positive: methylation level greater than 90%.

[0056] AS positive: methylation level is approximately less than 10%.

[0057] In a sixth aspect, the present invention provides a system for rapid diagnosis of PWS / AS, comprising the following modules:

[0058] (a) a collection module, which is used to provide genomic DNA to be detected;

[0059] (b) a methylation conversion module, which is used to perform methylation conversion on the genomic DNA to be detected in step (a) to obtain converted genomic DNA;

[0060] (c) an amplification module, which is used to perform multiplex MS-PCR amplification using the converted genomic DNA of step (b) as a template, using the primer set according to the first aspect and the primer set for quality control genes in the non-methylated region and the primer set for quality control genes in the methylated region according to the second aspect;

[0061] (d) a library construction module, which is used to construct an NGS library;

[0062] (e) A sequencing molecular module, which is used for NGS library sequencing and analysis of the average methylation ratios in the 15q11.2-q13 region of SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI13, and interpretation of the results.

[0063] In one embodiment, the genomic DNA in the collection module is genomic DNA from a whole blood sample, and preferably a commercial DNA extraction kit can be used.

[0064] In one embodiment, the methylation conversion module converts methylation into methylase conversion; preferably, a commercial enzyme conversion kit can be used.

[0065] In one embodiment, the amplification module comprises the kit according to the third aspect.

[0066] In one embodiment, the amplification module, the reaction system of multiplex MS-PCR amplification is a 50 μL reaction system: 10×LA PCR Buffer II (Mg 2+ plus) 5.0μL, dNTP Mixture 8.0μL, TaKaRa LA Taq 0.5μL, Primer Mix 5.0μL, template DNA 5.0μL, make up to 50.0μL with sterile deionized water;

[0067] Among them, 10×LA PCR Buffer II (Mg 2+ Mg in plus 2+ The concentration is 25mM; in the dNTP Mixture, the concentration of each dNTP is 2.5mM; the concentration of the enzyme in TaKaRa LA Taq is 5U / μL, and the storage solution includes the following components: 20mMTris-HCl (PH8.0), 100mM potassium chloride, 0.1mM EDTA, 1mM DTT, 0.5% Tween-20, 0.5% NP-40, 50% glycerol; Primer Mix includes SEQ ID No.1-SEQ ID No.10, and the concentration of each primer is 2.5pmol / μL.

[0068] In one embodiment, the amplification module, the reaction program of multiplex MS-PCR amplification is: 95°C 3min, 95°C 30sec, 55°C 30sec (-0.5°C / cycle), 72°C 1min, 10 cycles; 95°C 30sec, 55°C 30sec, 72°C 1min, 25 cycles; 72°C 5 min, 4°C hold.

[0069] In one embodiment, in the amplification module, the multiplex MS-PCR amplification products can be further purified before the next step of reaction; the purification is performed using DNA purification magnetic beads.

[0070] In one embodiment, the library construction module includes reagents for constructing an NGS library, and the reagents for constructing an NGS library include adapters, ligases, DNA purification magnetic beads, library PCR primers, and library PCR reaction buffer.

[0071] In one embodiment, the NGS library sequencing is performed using a second-generation high-throughput sequencer; different sequencing platforms can be adapted according to the type of NGS library, and the amount of sequencing data on each sample library is 1.0G. Further selected from BGI DNBSEQ-T7 high-throughput sequencing.

[0072] In one embodiment, after performing NGS library sequencing and analyzing the average methylation ratios in the SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI13 regions of the 15q11.2-q13 region by a sequencing analysis module, the results are interpreted:

[0073] Interpretation criteria:

[0074] Negative samples: methylation level is 50% ± 20%.

[0075] PWS positive: methylation level greater than 90%.

[0076] AS positive: methylation level is approximately less than 10%.

[0077] Compared with the prior art, the present invention has the following advantages and effects:

[0078] The present invention uses methylation-specific polymerase chain reaction (MS-PCR) combined with NGS sequencing technology to detect the methylation level of the SNRPN gene in the 15q11.2-q13 region of chromosome 15. The detection method can be used for rapid molecular diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) epigenetic inheritance. The method is easy to operate, has low detection cost, good detection result accuracy, high detection sensitivity and specificity, and high detection throughput, and is suitable for clinical promotion and application. The detection results of this method can accurately distinguish the abnormal methylation level of the chromosome region 15q11.2-q13, and can be used for rapid auxiliary diagnosis of PWS syndrome and AS syndrome. DETAILED DESCRIPTION

[0079] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The materials involved in the following examples can be obtained from commercial channels unless otherwise specified. The methods described are conventional methods unless otherwise specified.

[0080] In the embodiments of the present invention, the amplification primers (SEQ ID No. 1-SEQ ID No. 10) of SNRPN-CGI1, SNRPN-CGI2, SNRPN-CGI3, KLF4, and RHOG genes were synthesized by Guangzhou Aiki Biotechnology Co., Ltd. The genomic DNA methylation enzymatic conversion reagent EpiArt DNA Enzymatic Methylation Kit (EM301) was purchased from Nanjing Novogene Biotechnology Co., Ltd. NGS library preparation reagents and magnetic bead purification reagents were purchased from Jiajian (Guangzhou) Bioengineering Technology Co., Ltd. The NGS library was sent to Beijing Geneplus Technology Co., Ltd. for high-throughput sequencing.

[0081] Example 1

[0082] This embodiment is a method for operating an epigenetic molecular diagnosis experiment for Prader-Willi syndrome (PWS) and Angelman syndrome (AS).

[0083] 1. Extraction of sample genomic DNA:

[0084] Whole blood samples were collected from subjects with Prader-Willi syndrome / Angelman syndrome of different etiologies and normal subjects, and genomic DNA was extracted using a commercial kit, and the extracted DNA was stored at −20°C and stored at −80°C for long-term storage.

[0085] 2. Genomic DNA methylation conversion

[0086] Refer to the instructions of EpiArt DNA Enzymatic Methylation Kit (EM301) for genomic DNA methylation enzymatic conversion, and use 100ng of genomic DNA as input for enzymatic methylation DNA conversion. The cytosine that has not undergone methylation modification on the genome is converted to uracil. The methylated DNA is stored at -20℃, and stored at -80℃ for long-term storage.

[0087] 3. MS-PCR Amplification

[0088] Using the methylated genomic DNA as a template, multiple PCR amplification was performed using amplification primers (SEQ ID No. 1-SEQ ID No. 10) of the SNRPN-CGI1, SNRPN-CGI2, SNRPN-CGI3, KLF4, and RHOG genes, respectively. The reaction system was prepared according to the following PCR amplification system:

[0089]

[0090] Among them, 10×LA PCR Buffer II (Mg 2+ Mg in plus 2+ The concentration is 25mM; in the dNTP Mixture, the concentration of each dNTP is 2.5mM; the concentration of the enzyme in TaKaRa LA Taq is 5U / μL, and the storage solution includes the following components: 20mMTris-HCl (PH8.0), 100mM potassium chloride, 0.1mM EDTA, 1mM DTT, 0.5% Tween-20, 0.5% NP-40, 50% glycerol; Primer Mix includes SEQ ID No.1-SEQ ID No.10, and the concentration of each primer is 2.5pmol / μL.

[0091] Mix the prepared reaction system, centrifuge briefly, transfer to a PCR instrument, and amplify according to the following PCR reaction program:

[0092]

[0093] After PCR, 2 μL of PCR product was taken from each sample and tested by 1.5% agarose gel electrophoresis (180V, 17min) to confirm the fragment size. The sizes of the 5 fragments (SNRPN-CGI1, SNRPN-CGI2, SNRPN-CGI3, KLF4, and RHOG genes) were 283 bp, 378 bp, 308 bp, 304 bp, and 206 bp, respectively. The product does not need to be fragmented and can be directly used for NGS library preparation. Then PCR product purification was performed according to the following steps:

[0094] (1) Take out the purified magnetic beads 30 minutes in advance and place them at room temperature. Vortex and mix thoroughly before use.

[0095] (2) Add 90 μL of purified magnetic beads (1.8×) to the amplified product tube, incubate at room temperature for 5 min, then centrifuge briefly. Place the PCR tube on a magnetic rack until the solution becomes clear.

[0096] (3) Discard the supernatant, add 200 μL 80% ethanol, and let stand for 30 seconds;

[0097] (4) Discard the supernatant, add 200 μL 80% ethanol, and let stand for 30 seconds;

[0098] (5) Carefully discard the supernatant and absorb as much ethanol as possible, but be careful not to absorb the magnetic beads at the bottom;

[0099] (6) Keep the PCR tube on the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 min until the surface of the beads is non-reflective and free of cracks (be careful not to over-dry, otherwise it will cause DNA loss);

[0100] (7) Add 32 μL of ddH2O and mix by pipetting. Incubate at room temperature for 5 min. Place on a magnetic rack (about 2 min) until the solution becomes clear. Carefully transfer 30 μL of the supernatant to a new tube (do not touch the magnetic beads).

[0101] The concentration of each purified PCR product was measured using a Qubit 4.0 fluorometer. The purified PCR product can be stored stably at 4°C for one week. For long-term storage, place it at -20°C and avoid repeated freezing and thawing.

[0102] 4. NGS Library Preparation

[0103] 4.1 End repair

[0104] Determine the concentration of the PCR purified product, take 100 ng of each sample and add water to 50 μL, and add End Prep Mix 4 to each tube for end repair according to the following requirements.

[0105]

[0106] Use a pipette to gently pipette to mix (do not oscillate to mix), and centrifuge briefly; incubate in a PCR instrument: heat cover at 105°C.

[0107]

[0108] 4.2 Connector connection

[0109] Add the following Adapter Ligation Reaction Solution to each End Preparation product:

[0110]

[0111] Use a pipette to gently mix (do not oscillate), centrifuge briefly, and incubate in a PCR instrument: heat cover at 50°C.

[0112]

[0113] 4.3 Purification of ligation product

[0114] Follow the steps below to purify the ligation product:

[0115] (1) Take out the purified magnetic beads 30 minutes in advance and place them at room temperature. Vortex and mix thoroughly before use.

[0116] (2) Add 60 μL of purified magnetic beads to the amplified product tube, incubate at room temperature for 5 min, then centrifuge briefly. Place the PCR tube on a magnetic rack until the solution becomes clear.

[0117] (3) Discard the supernatant, add 200 μL 80% ethanol, and let stand for 30 seconds;

[0118] (4) Discard the supernatant, add 200 μL 80% ethanol, and let stand for 30 seconds;

[0119] (5) Carefully discard the supernatant and absorb as much ethanol as possible, but be careful not to absorb the magnetic beads at the bottom;

[0120] (6) Keep the PCR tube on the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 min until the surface of the beads is non-reflective and free of cracks (be careful not to over-dry, otherwise it will cause DNA loss);

[0121] (7) Add 22 μL of ddH2O and mix by pipetting. Incubate at room temperature for 5 min. Place on a magnetic rack (about 2 min) until the solution becomes clear. Carefully transfer 20 μL of the supernatant to a new tube (do not touch the magnetic beads).

[0122] Note: The samples can be stored stably at 4°C for one week. For long-term storage, place at -20°C and avoid repeated freezing and thawing.

[0123] 4.4 Library PCR amplification

[0124] Set up the PCR system and mix according to the following standards:

[0125]

[0126] Use a pipette to mix gently (do not oscillate), centrifuge briefly, and run the following program on the PCR instrument: heat the lid at 105°C.

[0127]

[0128] 4.5 Library PCR product purification

[0129] (1) Take out the purified magnetic beads 30 minutes in advance and place them at room temperature. Vortex and mix thoroughly before use.

[0130] (2) Add 45 μL of purified magnetic beads (0.9X) to the amplified product tube, incubate at room temperature for 5 min, then centrifuge briefly. Place the PCR tube on a magnetic rack until the solution becomes clear.

[0131] (3) Discard the supernatant, add 200 μL 80% ethanol, and let stand for 30 seconds;

[0132] (4) Discard the supernatant, add 200 μL 80% ethanol, and let stand for 30 seconds;

[0133] (5) Carefully discard the supernatant and absorb as much ethanol as possible, but be careful not to absorb the magnetic beads at the bottom;

[0134] (6) Keep the PCR tube on the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 min until the surface of the beads is non-reflective and free of cracks (be careful not to over-dry, otherwise it will cause DNA loss);

[0135] (7) Add 32 μL of ddH2O and mix by pipetting. Incubate at room temperature for 5 min. Place on a magnetic rack (about 2 min) until the solution becomes clear. Carefully transfer 30 μL of the supernatant to a new tube (do not touch the magnetic beads).

[0136] Note: The samples can be stored stably at 4°C for one week. For long-term storage, place at -20°C and avoid repeated freezing and thawing.

[0137] 4.6 Library product quality inspection

[0138] The purified library product was subjected to 1.5% agarose gel electrophoresis and the Qubit instrument to determine the size and concentration of the amplified DNA fragment. The size of the amplified DNA fragment should be between 300bp and 500bp, and the next step of the experiment can be continued.

[0139] 5. NGS library sequencing

[0140] The constructed NGS library was sent to Beijing Geneplus Technology Co., Ltd. for BGI DNBSEQ-T7 high-throughput sequencing (NGS library circularization and DNB preparation were prepared by the sequencing service provider). The amount of sequencing data for each sample was 1.0G.

[0141] 6. Data Analysis of Sequencing Results

[0142] The NGS sequencing data is analyzed using Bismark software. The process and principle of Bismark analysis of methylation NGS results are as follows:

[0143] Principle: BISMA analysis is based on the bisulfite conversion method, which converts unmethylated cytosine (C) to uracil (U). During the subsequent PCR amplification process, U will be replaced by thymine (T), while methylated C will not be affected.

[0144] process:

[0145] 1. Genome index construction: Bisulfite treatment is performed on the reference genome to simulate conversion (C->T and G->A), and an index is created using tools (such as bowtie2).

[0146] 2. Sequence alignment: Perform Bisulfite conversion on the sequenced reads and align them with the converted genome index, and output the alignment results (BAM format) and statistical reports.

[0147] 3. Methylation site extraction (optional): Use the alignment results to extract methylation information, including the location of the methylation site, methylation level, etc., and perform in-depth analysis as needed.

[0148] During the analysis, the gene sequences of SNRPN-CGI1, SNRPN-CGI2, SNRPN-CGI13, KLF4, and RHOG (taken from hg19) were treated with Bisulfite as the reference genome. The average methylation ratio of the KLF4 region obtained by Bismark analysis was used as the non-methylated internal reference (the average methylation ratio of this region in normal people is less than 5%), and the average methylation ratio of the RHOG region obtained by Bismark analysis was used as the methylated internal reference (the average methylation ratio of this region in normal people is greater than 90%). The methylation ratios of all methylated sites in the regions of SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI13 were counted and the average value was calculated. The results were interpreted based on this average value.

[0149] Interpretation criteria:

[0150] Negative samples: methylation level is 50% ± 20%.

[0151] PWS positive: methylation level greater than 90%.

[0152] AS positive: methylation level is approximately less than 10%.

[0153] Example 2

[0154] This embodiment is a clinical sample detection of an epigenetic molecular diagnostic method for Prader-Willi syndrome (PWS) and Angelman syndrome (AS).

[0155] Eight clinically positive samples suspected of PWS, two clinically positive samples suspected of AS, and eight negative samples were collected, and epigenetic molecular detection and analysis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) were performed according to the detection method flow in Example 1. As a result, 8 PWS positives, 2 AS positives, and the other 8 negatives were detected in 18 samples. The specific test results are shown in the table below. The test results were further compared with the clinical test results of the samples, and the test results were 100% consistent.

[0156]

[0157] Note: PWS Deletion is paternal deletion type PWS, and PWS Disomy is maternal uniparental disomy (UPD).

[0158] Therefore, the results of the detection by the method of the present invention, compared with the clinical test method, both the specificity and sensitivity reached 100%. Moreover, among the 18 samples, 8 samples were determined to be PWS positive, 2 samples were AS positive, and 8 samples were negative by the method of the present invention.

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a primer set in preparing a kit for rapid diagnosis of PWS / AS, characterized in that: The PWS / AS rapid diagnosis includes the steps of testing the sample by NGS sequencing; The primer set includes the following primers: Two specific primers used to amplify the SNRPN-CGI1 gene: Primer 1: 5′-gAgggAgTTgggATTTTTgTA-3′; Primer 2: 5′-AAATCCTAAAAACTTAAAATATCTAATAAA-3′; Two specific primers used to amplify the SNRPN-CGI2 gene: Primer 1: 5′-AATTAgATTYggAATgTTTAgAgg-3′; Primer 2: 5′-CAATAATCACTATTATACACCTACCTA-3′; Two specific primers used to amplify the SNRPN-CGI3 gene: Primer 1: 5′-TTAAAgTgTTgTTTgggAAAggA-3′; Primer 2: 5′-AATACCTAACCTCTAACCACATTCC-3′; Two specific primers used to amplify the KLF4 gene: Primer 1: 5′-gAgAATAAAgTTTAggTTTAggAgAT-3′; Primer 2: 5′-ATAACAACAAAAAACCACCACTAAC-3′; Two specific primers used to amplify the RHOG gene: Primer 1: 5′-TATATTTTTggATgTTATTTgTgT-3′; Primer 2: 5′-TAAACCTAAACCTATAAAACACTAC-3′.

2. The use according to claim 1, characterized in that: The PWS / AS rapid diagnosis includes the following steps: (1) Provide genomic DNA to be tested; (2) methylating the genomic DNA to be detected in step (1) to obtain converted genomic DNA; (3) using the transformed genomic DNA of step (2) as a template, and performing multiplex MS-PCR amplification using the primer set according to claim 1; (4) Construction of NGS library; (5) Perform NGS library sequencing and analyze the average methylation ratios of SNRPN-CGI1, SNRPN-CGI2, and SNRPN-CGI13 in the 15q11.2-q13 region, and interpret the results.

3. A device for rapid diagnosis of PWS / AS, characterized in that: The device comprises a kit comprising the primer set described in claim 1 and a kit for constructing an NGS library.

4. The device according to claim 3, characterized in that: The kit for constructing an NGS library comprises an end repair enzyme, a linker, a ligase, DNA purification magnetic beads, a library PCR primer and a library PCR reaction buffer.

5. A system for rapid diagnosis of PWS / AS, characterized in that: Includes the following modules: (a) a collection module, which is used to provide genomic DNA to be detected; (b) a methylation conversion module, which is used to perform methylation conversion on the genomic DNA to be detected in step (a) to obtain converted genomic DNA; (c) an amplification module, which is used to perform multiplex MS-PCR amplification using the converted genomic DNA of step (b) as a template and the primer set described in claim 1; (d) a library construction module, which is used to construct an NGS library; (e) A sequencing analysis module, which is used for NGS library sequencing and analyzing the average methylation ratios in the SNRPN-CGI1, SNRPN-CGI2 and SNRPN-CGI13 regions of the 15q11.2-q13 region, and interpreting the results.

6. The system according to claim 5, characterized in that: The genomic DNA is genomic DNA from a whole blood sample; The library construction module includes reagents for constructing an NGS library, and the reagents for constructing an NGS library include a linker, a ligase, DNA purification magnetic beads, a library PCR primer, and a library PCR reaction buffer; The NGS library sequencing is performed using a second-generation high-throughput sequencer; different sequencing platforms are adapted according to the type of NGS library.

7. The system according to claim 5 or 6, characterized in that: The sequencing analysis module is used to perform NGS library sequencing and analyze the average methylation ratios in the SNRPN-CGI1, SNRPN-CGI2 and SNRPN-CGI13 regions of the 15q11.2-q13 region, and the results are interpreted: Interpretation criteria: Negative samples: methylation level is 50% ± 20%; PWS positive: methylation level greater than 90%; AS positive: methylation level is less than 10%.

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