Primer set, kit and application for detecting multiple mutations of GBA1 gene and its pseudogene GBAP1 in Gaucher disease

Abstract

The present invention provides a primer set, a kit and their applications for detecting multiple mutations of the GBA1 gene and its pseudogene GBAP1 in Gaucher disease. Specifically, the present invention provides a primer set for simultaneously amplifying or detecting multiple mutations of the GBA1 gene and its pseudogene GBAP1, the primer set includes GBA-F primer, GBA-R primer and GBAP-R primer and further includes Gap-F primer. The present invention also provides a kit containing the above primer set. Based on the specific combination of homologous region multiplex long fragment GAP PCR amplification and single molecule long high-throughput sequencing, the present invention can achieve highly specific, accurate and rapid simultaneous detection of multiple samples related to GD GBA1 pathogenic genes and their pseudogenes GBAP1 multiple mutations.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly relates to a primer set, a kit and an application for detecting Gaucher disease-related GBA1 genes and their pseudogenes GBAP1 with multiple mutations. Background Art

[0002] Gaucher disease (GD), a rare autosomal recessive genetic disease, is caused by GBA1 gene mutations. The characteristics of Gaucher disease are hepatosplenomegaly, cytopenia, sometimes severe bone involvement, and in some forms, nerve damage 1 . GD can be divided into 3 types according to clinical manifestations. Type 1 GD (GD1, chronic non-neuropathic, proportion: 90%-95%) is the most common phenotypic form of this disease. Its clinical manifestations are variable, ranging from asymptomatic throughout life to early-onset forms that appear in childhood. The overall average age of onset of patients is 20.4 years 2 . GD1 usually affects the quality of life but rarely endangers life. Common phenotypes include fatigue, growth retardation and delayed puberty, hepatosplenomegaly, gallstones, bone involvement, etc. 2,3,4 . Type 2 GD (GD2, acute neuropathic, proportion <5%) is characterized by early and severe phenotypic symptoms starting in infants aged 3-6 months, brainstem involvement (i.e., abnormal eye movements, spastic state, hypotonia) and seizures, as well as life-threatening systemic symptoms such as respiratory distress and aspiration pneumonia, and the life expectancy is less than 2 years 5 . Type 3 GD (GD3, chronic neuropathic, proportion 5%), the phenotype of GD3 is also very heterogeneous, mainly manifested in hepatosplenic involvement, cardiac involvement (valve calcification), aortic calcification, etc., and in terms of the nervous system, it includes progressive myoclonic epilepsy, cerebellar ataxia, spasticity or dementia 6,7,8 . It has a significant negative impact on the quality of life related to children's health. In addition to the above three types, fetal GD is the rarest (<1%) and the most severe form of the disease. It usually presents as fetal hydrops, hepatosplenomegaly, ichthyosis, joint contractures, facial deformities, and death usually occurs in utero or shortly after birth 9 . The incidence of GD in the general population is about 1 / 40,000 to 1 / 60,000, but it can reach 1 / 800 in Ashkenazi Jews 10 . Due to clinical heterogeneity, patients are often misdiagnosed and the time to obtain an accurate diagnosis is delayed. The diagnosis of GD patients is often delayed for up to 10 years. Early diagnosis is very important for starting appropriate treatment, preventing complications and disease progression 11 .

[0003] GBA1The gene is located on the long arm of chromosome 1 (1q21) and contains 11 exons. There is a highly homologous pseudogene at the same locus (16 kb downstream), resulting in GBAP1 recombination events between GBA1 and GBAP1 . The sites of these recombination events are variable, ranging from intron 2 to exon 11 12 . To date, approximately 400 pathogenic mutations have been identified in the GBA1 gene. These include substitutions, insertions, deletions, and complex alleles. The most common mutations are missense mutations, among which the point mutations c.1226A>G (N370S) and c.1448T>C (L444P) are the most frequent. However, the N370S mutation is rarely found in Chinese and Japanese patients, and in Asian ethnic groups, c.1448T>C (L444P / L483P) and c.754T>A (F252I) are more prevalent 13 .

[0004] Initially, laboratories used PCR-based screening techniques to identify a limited number of known mutations. In patients with type GD1 of Ashkenazi Jewish descent, this was a fairly effective method 14 . However, especially in patients with neurological GD, when based on limited mutations, more rare pathological variants may be missed, leading to false negatives. DNA sequencing using the Sanger method has been the gold standard for mutation identification in GD patients for the past 30 years, but due to the limitations of Sanger sequencing, it is not suitable for systematic analysis of GBA1 15 .

[0005] The emergence, increased availability, and reduced cost of NGS have become important factors for its more widespread use in establishing challenging diagnoses. The more extensive inclusion of NGS in genotype GD patients has led to the discovery of many new GBA1 mutations. However, when new GBA1 variants are discovered, these results may be difficult to interpret. In addition, recombinant alleles generated by genomic recombination with highly homologous GBA1 pseudogenes may be difficult to detect by this method 16 . Therefore, in addition to NGS analysis, at least 30 regions of this gene were analyzed by Sanger sequencing. Without Sanger sequencing support, many studies using NGS technology did not report the presence of recombinant mutations, which increased the likelihood of underestimating the existence of these alleles 15 .

[0006] In recent years, with the emergence and development of SMRT sequencing and Nanopore sequencing, long-read sequencing of several kb can be easily achieved, completing the sequencing of​GBA1 Sequencing of gene regions 17 . However, currently GBA1 The long-fragment enrichment systems for genes only focus on GBA1 gene regions. Such an enrichment method has certain defects and cannot detect all GBA1 and GBAP1 types of structural variations formed between them, resulting in the omission of some structural variation types 18 . The existing enrichment methods also exclude the detection of GBAP1 genes. Some studies have shown that GBAP1 may act as a ceRNA to regulate GBA1 gene expression 19 . None of the existing systems simultaneously enrich GBA1 and GBAP1 genes. Although it reduces the amplification difficulty and avoids non-specific amplification in homologous regions, it can more precisely enrich functional genes; however, due to the lack of enrichment of pseudogenes, the detection system becomes more complex and cannot comprehensively screen and display the mutation situations in this region.

[0007] The existing technologies can achieve GBA1 detection of some structural variations and point mutations, but the detection has the following limitations: It cannot achieve simultaneous detection of all point mutation classes and structural variation types of genes in the same system; due to GBA1 the high homology between GBA1 and GBAP1 , it may affect the design of Sanger sequencing primers and the sequencing results of NGS, leading to a certain degree of false negatives or false positives; when there are two or more mutations at the same gene locus simultaneously, the existing methods cannot distinguish cis or trans mutations, and whether the mutations have a linkage effect; the existing GBA1 long-fragment amplification technology only enriches for GBA1 gene regions. If GBA1 and GBAP1 undergo non-allelic interaction due to homologous recombination, it cannot be detected; the existing technologies ignore the detection of non-functional genes GBAP1 , and cannot achieve a "comprehensive version" detection of the GBA1 region; since traditional methods require a combination of multiple technologies, it leads to labor intensity, higher requirements for sample quality, longer gene diagnosis time, and high detection costs. Summary of the Invention

[0008] As mentioned above, the existing long-fragment amplification technology usually only enriches for GBA1 gene regions and excludes the detection of GBAP1The amplification of genes, although reducing the amplification difficulty and avoiding non-specific amplification of homologous regions, may lead to the inability to detect the interaction between non-allelic genes caused by homologous recombination. In view of this, the present invention provides a method based on multiplex long-fragment GAP PCR amplification of homologous regions and single-molecule sequencing to detect GD-related GBA1 genes and their pseudogenes GBAP1 for various mutations.

[0009] Based on the special design and setting of the primer set, the present invention can not only restore the variation of the entire GBA1 and GBAP1 gene regions, but also overcome the amplification preference problem of simultaneous enrichment of GBA1 and GBAP1 gene homologous regions, reduce the problem of easy background generation in homologous regions, and achieve multiplex long-fragment GAP PCR amplification of homologous regions in a single reaction tube, while amplifying the GBA1 gene fragments for detecting exon SNVs and InDels, the GBA1 deletion and fusion detecting GBAP1 pseudogene fragments and Gap fragments for detecting large fragment deletions. Combining the characteristics of long read lengths of the single-molecule sequencing platform, the present invention can accurately, rapidly and high-throughput detect GBA1 genes and their pseudogenes GBAP1 for various mutations. The method involved in the present invention is simple to operate, the multiplex long-fragment GAP PCR and single-molecule sequencing library have reliable quality and strong repeatability, which is conducive to the application of single-molecule sequencing technology in clinical detection.

[0010] The purpose of the present invention is to solve the problems in the current stage GBA1 where the detection of pathogenic genes is incomplete, the gene diagnosis detection methods are cumbersome, and the single variation types detected by different methods lead to missed and misdiagnosed cases clinically. By simultaneously amplifying multiple fragments of GD-related GBA1 pathogenic genes and their pseudogenes GBAP1 by multiplex PCR and preparing a single-molecule sequencing library, the goal of comprehensively, accurately and rapidly detecting multiple mutations of GD-related genes GBA1 and their pseudogenes GBAP1 is achieved.

[0011] On the one hand, the present invention provides a primer set for simultaneously amplifying or detecting GBA1 genes and their pseudogenes GBAP1Multiple mutations, and the primer set comprises a GBA-F primer, a GBA-R primer, and a GBAP-R primer; wherein the nucleotide sequence of the GBA-F primer is as shown in SEQ ID NO: 1-2, the nucleotide sequence of the GBA-R primer is as shown in SEQ ID NO: 5-7, and the nucleotide sequence of the GBAP-R primer is as shown in SEQ ID NO: 3-4. In some embodiments, the primer set further comprises a Gap-F primer, wherein the nucleotide sequence of the Gap-F primer is as shown in SEQ ID NO: 8-11. All primer positions are as Figure 1 shown.

[0012] In some embodiments, the GBA1 gene and its pseudogene GBAP1 multiple mutations include GBA1 SNVs and InDels on the exons of the gene, GBA1 deletions and fusions caused by recombination with GBAP1 the gene and their corresponding GBA1 gene copy number variations, GBA1 large fragment deletion variations within 20 kb upstream. In some embodiments, the GBA1 mutations of the gene include at least 640 mutations as shown in Table 1.

[0013]

[0014]

[0015]

[0016]

[0017]

[0018] In some preferred embodiments, the primer set is used for homologous region multiplex long fragment GAP PCR amplification, and the GBA1, GBAP1 gene fragment and the Gap fragment are amplified simultaneously in one reaction system. Therefore, the primer set can simultaneously detect GBA1 SNVs and InDels on the exons of the GBA1 gene, GBAP1 deletions and fusions caused by recombination with the GBA1 gene and their corresponding GBA1 gene copy number variations,

[0019] large fragment deletion variations within 20 kb upstream. The point mutations and structural variations at the gene loci described herein can be queried in LOVD, ClinVar, and the references, and some mutation information is shown in Table 1.In some embodiments, the amplification product of the primer set is 10 kb - 18 kb. In some preferred embodiments, if there are SNPs at the primer positions, degenerate base primers are used.

[0020] In some embodiments, the primer set is used to detect whether multiple mutations within the amplified product fragment are linked.

[0021] In some embodiments, the 5'-end of the primers in the primer set further comprises a DNA barcode for differentiating different samples. In some preferred embodiments, the barcodes at the 5'-ends of the F and R primers can be the same or different, and those skilled in the art can select according to needs. In some preferred embodiments, the length of the DNA barcode is 5 - 50 nt.

[0022] On the other hand, the present invention provides a kit for simultaneously amplifying or detecting GBA1 a gene and its pseudogene GBAP1 multiple mutations, which comprises the following reagents: (1) reagents for homologous region multiplex long fragment GAP PCR amplification; (2) reagents for constructing a single molecule sequencing library; wherein the reagents for homologous region multiplex long fragment GAP PCR amplification comprise the primer set as described in the present invention.

[0023] In some embodiments, the GBA1 gene and its pseudogene GBAP1 multiple mutations comprise GBA1 SNVs and InDels on the gene exon, GBA1 deletions and fusions caused by recombination with the GBAP1 gene and their corresponding GBA1 gene copy number variations, GBA1 large fragment deletion variations within 20 kb upstream. In some preferred embodiments, the mutations of the GBA1 gene comprise at least 640 mutations as shown in Table 1.

[0024] In some embodiments, the reagents for homologous region multiplex long fragment GAP PCR amplification simultaneously amplify the GBA1, GBAP1 gene fragment and the Gap fragment in one reaction system. In some embodiments, the reagents for homologous region multiplex long fragment GAP PCR amplification further comprise a DNA polymerase and a reaction buffer. For the kit, the PCR amplification product can be purified or not purified before the next reaction, and those skilled in the art can select according to needs.

[0025] In some embodiments, the reagents for constructing a single molecule sequencing library comprise adapters, ligases, DNA purification magnetic beads, reaction buffers and exonucleases.

[0026] On the other hand, the present invention provides a system for simultaneously amplifying or detecting GBA1 a gene and its pseudogene GBAP1 multiple mutations, which includes the following modules: (1) a collection module: for obtaining and preparing a subject sample; (2) an amplification module: for performing homologous region multiplex long fragment GAP PCR amplification on the sample; (3) a library construction module: for constructing a single molecule sequencing library; (4) a sequencing module: for sequencing and analyzing the mutation types; wherein the homologous region multiplex long fragment GAP PCR amplification in the collection module is carried out using the primer set as described in the present invention or the kit as described in the present invention.

[0027] In some embodiments, the GBA1 gene and its pseudogene GBAP1 multiple mutations include GBA1 SNVs and InDels on the exons of the gene, GBA1 deletions and fusions caused by recombination with GBAP1 the gene and their corresponding GBA1 gene copy number variations, GBA1 large fragment deletion variations within 20 kb upstream. In some embodiments, the mutations of the GBA1 gene include at least 640 mutations shown in Table 1.

[0028] In some embodiments, the sample is selected from biological samples or gDNA extracted from the biological samples. Preferably, the biological samples include cultured cell lines, blood, amniotic fluid, chorionic villi, gametes, blastocyst cells, synovial fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile, and pancreatic fluid.

[0029] In some embodiments, the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0030] In a preferred embodiment, single molecule gene sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0031] In a specific embodiment, the PacBio library adapter ligation can be carried out by blunt-end ligation or TA ligation.

[0032] In a specific embodiment, the PacBio common blunt-end linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 12), which forms a blunt-end stem-loop structure adaptor by annealing. DNA (Barcode) with different sequences of 5-50 nt can be added to the stem to form different Barcode-containing adaptors. PacBio libraries with different Barcodes can be mixed together for sequencing.

[0033] In a specific embodiment, the PacBio common TA linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 13), which forms a blunt-end stem-loop structure adaptor by annealing. DNA (Barcode) with different sequences of 5-50 nt can be added to the stem to form different Barcode-containing adaptors. PacBio libraries with different Barcodes can be mixed together for sequencing.

[0034] In one embodiment, the PacBio adaptor can be with or without a Barcode. Preferably, the PacBio adaptor has a Barcode designed by Pacific Biosciences or a self-designed Barcode, which can be selected by those skilled in the art according to needs.

[0035] In a preferred embodiment, the PacBio library is matched with the sequencing platform of Pacific Biosciences.

[0036] In a preferred embodiment, the reagents for constructing the Nanopore library include end repair enzyme, adaptor, ligase, DNA purification magnetic beads, 80% ethanol, and reaction buffer.

[0037] In one embodiment, the ligation of the Nanopore library adaptor can be carried out by blunt-end ligation or TA ligation.

[0038] In one embodiment, the Nanopore adaptor can be with or without a Barcode. Preferably, the Nanopore adaptor has a Barcode designed by ONT or a self-designed Barcode, which can be selected by those skilled in the art according to needs.

[0039] In a preferred embodiment, the Nanopore library is matched with the sequencing platform of ONT.

[0040] The excellent technical effects of the primer set, kit, system, and method provided by the present invention mainly lie in the following aspects:

[0041] (1) Wide detection range. The present invention can simultaneously detect all point mutations and InDels on the exons of GD-related genes that have been studied and discovered so far, including more than 640 in total, and can distinguish the cis-trans relationships of all mutations on the same gene. GBA1 And gene recombination-induced deletions and fusions, GBA1 and GBAP1 large fragment deletion variations within 20 kb upstream, and the corresponding GBA1 gene copy number increases and decreases for these deleted and fused genes, and determine the accurate GBA1 positions of fusions and deletions. Since this system simultaneously enriches GBA1 genes, it can more accurately restore the variant situation of the entire GBAP1 gene region, providing convenience for subsequent scientific research and screening work. GBA1 and GBAP1 gene region, providing convenience for subsequent scientific research and screening work.

[0042] (2) Single-tube detection of multiple mutation types. Traditional methods require a detection system to be set up for each mutation type, while the present invention overcomes the GBA1 and GBAP1 amplification preference problem of co-enriching homologous regions of genes, reducing the problem of easy background generation in homologous regions, and achieving the simultaneous detection of multiple mutations in a reaction primer system, including SNV, Indel, gene recombination, and copy number variation.

[0043] (3) Low false detection and missed detection rates. The most common method for detecting point mutations in the currently commonly used GD pathogenic genes GBA1 is PCR + Sanger. Due to the GBA1 gene being huge, the existence of pseudogenes GBAP1 interference, and the limited detection range of Sanger and the cumbersome detection experiment, it is easy to cause missed diagnosis of pathogenic mutations and lead to false negative judgments. However, the method used in the present invention directly amplifies the entire exon regions of GBA1 and GBAP1 genes, all GBA1 related deletion fragments, GBA1 related fusion fragments, greatly reducing the risk of false detection and missed detection of patients, greatly improving the simplicity of GD gene diagnosis, and greatly reducing the time cost and labor cost of GD gene diagnosis.

[0044] (4) Sample diversification. The template for PCR can be peripheral blood, dried blood spots, or extracted genomic DNA, or it can be a human cell line or other specific tissues.

[0045] (5) High adaptability to sequencing platforms. The single-molecule sequencing library adapted by the amplification library construction method provided by the present invention is compatible with all commercial platforms based on SMRT sequencing and Nanopore sequencing.

[0046] (6) High-throughput detection. Single-molecule sequencing can achieve 384 types of Barcode adapters, and actually more types of Barcode adapters can be designed according to needs. Or use a dual-Barcode system with primers carrying Barcodes and adapters carrying Barcodes to achieve more combinations of Barcodes. The high-throughput characteristic of the single-molecule sequencing platform determines that high-throughput sample detection can be achieved.

[0047] (7) High precision. The dumbbell-shaped library of the SMRT library can be interpreted multiple times during sequencing. After correction, the base accuracy of the sequencing result is greater than 99%. Moreover, the SMRT sequencing errors are random, and through sequencing depth correction, the base accuracy is greater than 99.9%. Therefore, gene mutations within the primer detection range can be accurately interpreted.

[0048] (8) Flexible detection time. The Nanopore platform can generate data within a few minutes, and data analysis can be started within a few minutes or hours according to the actual data volume requirements. When high requirements are placed on the detection timeliness, the Nanopore platform has a time advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of multiplex PCR primer design.

[0050] Figure 2 It is a DNA gel electrophoresis diagram of the sample amplified by the multiplex PCR method in Example 1.

[0051] Figure 3 It is GBA1 SMRT sequencing result diagram of a healthy person sample.

[0052] Figure 4 It is GBA1 SMRT sequencing result diagram of an E8-E11 deletion sample.

[0053] Figure 5 It is GBA1 SMRT sequencing result diagram of a point mutation sample. DETAILED DESCRIPTION OF THE INVENTION

[0054] The exemplary embodiments of the present invention are described in detail, which should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0055] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. For any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.

[0056] The sequences used in the present invention are shown in Table 2.

[0057]

[0058] Example 1: Amplifying GD-related gene mutations using different primer combinations with the long-fragment multiplex PCR method of the present invention

[0059] Obtain and prepare blood, dried blood spots, and genomic DNA samples using the collection module and perform long-fragment multiplex PCR amplification on the samples using the amplification module. Prepare the reaction system according to Table 3 below to amplify peripheral blood, dried blood spots, and genomic DNA samples:

[0060]

[0061] On a PCR instrument, perform pre-amplification according to the conditions shown in Table 4 below:

[0062]

[0063] After amplification is completed, take 5 μL of each sample and detect it on a 1% DNA gel. The results are as Figure 2 shown. Using different samples as templates, the GD-related GBA1 genes and their pseudogenes GBAP1 can all be effectively amplified.

[0064] Example 2: Constructing a SMRT sequencing library using the homologous-region multiplex long-fragment GAP PCR method related to the present invention

[0065] Prepare the reaction system according to Table 5 below to amplify peripheral blood samples with different types of GD-related GBA1 gene mutations:

[0066]

[0067] On a PCR instrument, perform pre-amplification according to the conditions shown in Table 6 below:

[0068]

[0069] After the amplification is completed, put the amplification product into a centrifuge, centrifuge at 10000 rpm for 20 min. After centrifugation, place it horizontally and statically, and take 4 mL of the supernatant and add it to a new tube.

[0070] Step 2: Construct a SMRT sequencing library

[0071] Use the library construction module to construct a single-molecule sequencing library, and use the sequencing module to perform sequencing and analyze the mutation types. Prepare the reaction system according to Table 7 below:

[0072]

[0073] On a PCR instrument, perform the reaction under the following conditions: 37ºC for 20 min; 25ºC for 15 min; 65ºC for 10 min. After the reaction is completed, add 0.5 mL of Exonuclease III (NEB, Cat#M0206L) and 0.5 mL of Exonuclease VII (NEB, Cat#M0379L), and continue to react at 37ºC for 1 hour. Purify twice with 0.6x AMpure PB magnetic beads (PacBio, Cat#100-265-900) according to the manufacturer's instructions, and finally elute the DNA with 10 mL of Elution Buffer. The obtained DNA eluate is the target SMRT sequencing library. Measure the DNA concentration on a Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216) using Qubit dsDNA HS reagent (ThermoFisher, Cat# Q32851). When there are multiple sample SMRT libraries, equal amounts of the libraries can be mixed together to prepare a mixed library.

[0074] Step 3: SMRT sequencing and analysis

[0075] According to the total concentration and molar concentration of the library, react an appropriate volume of the library with the binding reagent (PacBio, Cat#101-820-200) and the primer (PacBio, Cat#100-970-100) to prepare the final library that can be loaded onto the machine. Representative sequencing results are as Figures 3 - 5 shown, where Figure 3 is the IGV schematic diagram of the gene of a healthy person GBA1 and Figure 4 is GBA1 the IGV schematic diagram of homologous recombination variation Figure 5 and GBA1 is the IGV schematic diagram of gene point mutation.

[0076] Example 3: GBA1Detection and Verification of Gene Mutation Detection

[0077] Collect the peripheral blood genomic DNA of 15 subjects as 15 validation samples. Referring to Example 2, use the method (and kit) of the present invention to simultaneously detect multiple mutations at GD-related GBA1 gene loci. Use the method of PCR + Sanger sequencing to detect GBA1 point mutations of the gene. Compare the results obtained by the present invention with the control results. The results are shown in Table 8, and the results of 15 samples are completely consistent.

[0078]

[0079] Therefore, compared with the PCR + Sanger sequencing method, the specificity and sensitivity of the results detected by the method of the present invention both reach 100%. Moreover, the allelic position relationships of each SNV are clarified for all 4 samples.

[0080] It should be noted that although the above implementation cases have proved a series of features of the present invention, for researchers and technicians in the field, the reaction reagents, reaction conditions, etc. involved in multiplex PCR reactions and single-molecule sequencing library construction can be adjusted and changed according to specific needs using the idea of the present invention. Therefore, for those skilled in the art, within the scope of not departing from the concept and principle of the present invention, several simple substitutions can still be made, and these should all be included in the protection scope of the present invention.

[0081] References

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[10] Stirnemann J, Vigan M, Hamroun D et al. The French Gaucher’sdisease registry: Clinical characteristics, complications and treatment of562 patients. Orphanet J Rare Dis. 2012, 7, 77.

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[12] Tayebi N, Stubblefield BK, Park JK et al. Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease. Am J Hum Genet. 2003, 72, 519-534.

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[0095]

[14] Koprivica V, Stone DL, Park JK et al. Analysis and classification of 304 mutant alleles in patients with type 1 and type 3 Gaucher disease. Am J Hum Genet. 2000, 66, 1777-1786.

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Claims

1. A primer set for simultaneous amplification and detection in one reaction system GBA1 Genes and their pseudogenes GBAP1 Multiple mutations, the primer set comprises a GBA-F primer, a GBA-R primer, a GBAP-R primer and a Gap-F primer; The nucleotide sequence of the GBA-F primer is shown in SEQ ID NOs: 1-2, the nucleotide sequence of the GBA-R primer is shown in SEQ ID NOs: 5-7, the nucleotide sequence of the GBAP-R primer is shown in SEQ ID NOs: 3-4, and the nucleotide sequence of the Gap-F primer is shown in SEQ ID NOs: 8-11; It is stated GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large deletion variant within 20kb upstream. 2 . The primer set according to claim 1 , wherein the 5′ end of the primer in the primer set further comprises a DNA barcode.

3. The primer set according to claim 2, wherein the DNA barcodes are the same or different, and the length of the DNA barcodes is 5-50 nt.

4. A method for simultaneous amplification and detection in one reaction system GBA1 Genes and their pseudogenes GBAP1 A kit for multiple mutations, comprising the following reagents: (1) Reagents for multiple long-fragment GAP PCR amplification of homologous regions; (2) Reagents for constructing single-molecule sequencing libraries; The reagents for homologous region multiple long fragment GAP PCR amplification include the primer set as described in any one of claims 1 to 3; It is stated GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large deletion variant within 20kb upstream.

5. The kit according to claim 4, wherein the reagents for multiple long-fragment GAP PCR amplification of homologous regions further comprise a DNA polymerase and a reaction buffer.

6. The kit according to claim 4, wherein the reagents for constructing a single-molecule sequencing library comprise adapters, ligase, DNA purification magnetic beads, reaction buffer and exonuclease.

7. A simultaneous amplification and detection GBA1 Genes and their pseudogenes GBAP1 A system of multiple mutations, which includes the following modules: (1) Collection module: used to obtain and prepare subject samples; (2) Amplification module: used to perform multiple long-fragment GAP PCR amplification of homologous regions on the sample; (3) Library construction module: used to construct single-molecule sequencing libraries; (4) Sequencing module: used for sequencing and analyzing mutation types; The homologous region multiple long fragment GAP PCR amplification in the acquisition module is performed using the primer set described in any one of claims 1 to 3 or the kit described in any one of claims 4 to 6; It is stated GBA1 Genes and their pseudogenes GBAP1 Multiple mutations include GBA1 SNV and InDel on gene exons, GBA1 and GBAP1 Deletions and fusions caused by gene recombination and their corresponding GBA1 Gene copy number variation, GBA1 Large deletion variant within 20kb upstream.

8. The system according to claim 7, wherein the GBA1 The gene mutations include at least 642 mutations as shown in Table 1.

9. The system according to claim 7, wherein the sample is selected from a biological sample or gDNA extracted from the biological sample.

10. The system of claim 9, wherein the biological sample comprises cultured cell lines, blood, amniotic fluid, chorionic villi, gametes, blastocyst cells, joint fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile, and pancreatic fluid.

11. The system according to claim 7, wherein the sequencing module is selected from the PacBio sequencing platform of Pacific Biosciences or the Nanopore sequencing platform of ONT.

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