Primer group and kit for detecting Fabry disease GLA gene mutation and application of primer group and kit

Abstract

The invention relates to a primer group and a kit for detecting Fabry disease GLA gene mutation and application of the primer group and the kit. The primer group comprises one or more of a GLA-F1 / R1 primer pair, a GLA-F2 / R2 primer pair, a GLA-F3 / R3 primer pair, a GLA-F4 / R4 primer pair, a GLA-F5 / R5 primer pair, a GLA-F6 / R6 primer pair and a GLA-F7 / R7 primer pair. The kit further comprises a GLA-Gap primer. Based on a specific combination of long-fragment PCR amplification and third-generation high-throughput sequencing, the method can be used for simultaneously detecting various mutations related to the Fabry disease pathogenic gene GLA of a plurality of samples in a high-specificity, accurate and rapid manner.

Description

Technical Field

[0001] The present invention relates to a method for detecting Fabry disease (FD) by using a third-generation long-read sequencing platform. GLA A primer set and method for detecting multiple mutations of a gene, and a kit suitable for the method. Background Art

[0002] Fabry disease (FD, OMIM 301500) is a rare X-linked lysosomal disorder caused by a gene encoding α-galactosidase A (α-Gal A). GLA The disease is caused by pathogenic mutations in the gene. The deficiency of α-GaIA leads to the accumulation of glycosphingolipids (mainly Gb3) and their derivatives, globulosphingosine (lyso-Gb3), in cells, tissues and organs (including kidneys, heart, central and peripheral nervous systems) throughout the body, thus causing blood circulation disorders and further leading to heart and kidney failure and nervous system disorders and death.

[0003] FD is an X-linked lysosomal disorder caused by GLA Caused by pathogenic variants in genes. GLA The gene consists of 7 exons and is approximately 10 kb in length. GLA The gene cDNA encodes a precursor protein of approximately 50 kDa (429 amino acids) that is proteolytically cleaved into the lysosomal mature protein.

[0004] About 3,000 species have been discovered so far. GLA Gene mutations, 60% of which are missense mutations, followed by nonsense mutations, gene deletions, gene insertions and splice site mutations, which cause frame shifts or signal peptide abnormalities and change the α-GaIA polypeptide sequence; in addition, large chromosome fragments are deleted, resulting in the entire GLA Gene deletion is also one of the causes of FD.

[0005] The diagnosis of Fabry disease abroad is confirmed by a combination of biochemical and molecular testing. First, biochemical diagnostic methods use ELISA, immunoassays and other methods to measure the protein content and activity of α-GaIA in plasma or peripheral blood leukocytes, urine, tears and cultured skin fibroblasts. Biochemical methods can directly detect the content and activity of α-GaIA protein in the human body, but the disadvantage of this method is that it is not suitable for detecting female carriers; secondly, it is impossible to know the exact site of the α-GaIA gene mutation.

[0006] In molecular testing, first-generation sequencing (Sanger) and second-generation sequencing (NGS) can detect GLA Point mutations and insertions and deletions of genes; however, Sanger sequencing can detect GLA The point mutation throughput is low and unknown mutations cannot be detected; however, the capture or long-fragment amplification process in NGS requires GLA Gene construction NGS sequencing library can be detected GLA Point mutations in genes are not compatible with other genetic tests and cannot be detected GLA Cis-trans information of gene mutations; not detected in whole exome sequencing (WES) of NGS GLA The introns of the gene will therefore be missed. GLA Multiplex ligation-dependent probe amplification (MLPA) can detect GLA Large fragments of the gene are missing, but this method requires simultaneous detection GLA The operation is cumbersome and cannot detect large quantities of samples at different sites upstream and downstream of the gene.

[0007] Currently, methods based on biochemical testing, first-generation sequencing or second-generation sequencing, and MLPA can achieve GLA Gene point mutation and large fragment deletion detection, but the detection has the following main limitations: 1. It is impossible to detect all mutation types simultaneously in the same system; 2. Biochemical testing cannot detect female carriers and the specific mutation type; 3. First-generation sequencing can only detect known point mutations, and has low throughput, and cannot detect insertion and deletion types; 4. Next-generation sequencing can detect GLA Different types of genes need to be tested in combination with different library construction methods, which is cumbersome and costly; 5. MLPA can only detect a few known point mutations and cannot determine the exact site of large fragment deletions. Summary of the invention

[0008] In view of this, the present invention provides a method based on long-fragment PCR amplification and third-generation sequencing to detect multiple FD-related mutations. Long-fragment PCR amplification is achieved in one reaction tube. The method is used to detect GLA Point mutations in genes, GLA Insertions and deletions within genes, large deletions within the range of GLA-Gap primers. Combined with the characteristics of the third-generation sequencing platform with long read length measurement, accurate, rapid and high-throughput detection of FD-related gene mutations can be achieved. The method involved in the present invention is simple to operate, and the quality of long-fragment PC and third-generation library is reliable and highly reproducible, which is conducive to the application of third-generation sequencing technology in clinical detection.

[0009] The purpose of this invention is to solve the problem of FD pathogenic gene at present GLA Incomplete detection coverage, missed detection by specific NGS detection process, and NGS detection cannot distinguish the cis-trans structure of mutations. Amplify FD-related pathogenic genes by long-range PCR GLA The region and preparation of third-generation sequencing libraries achieve the goal of comprehensive, accurate and rapid detection of multiple FD mutations in multiple samples.

[0010] In a first aspect, the present invention provides a primer set for simultaneously amplifying multiple mutations of Fabry disease, wherein the primer set comprises one or more pairs of GLA-F1 / R1 primer pair, GLA-F2 / R2 primer pair, GLA-F3 / R3 primer pair, GLA-F4 / R4 primer pair, GLA-F5 / R5 primer pair, GLA-F6 / R6 primer pair, and GLA-F7 / R7 primer pair; wherein the nucleotide sequences of the GLA-F1 / R1 primer pair, the GLA-F2 / R2 primer pair, the GLA-F3 / R3 primer pair, the GLA-F4 / R4 primer pair, the GLA-F5 / R5 primer pair, the GLA-F6 / R6 primer pair, and the GLA-F7 / R7 primer pair are SEQ ID NOs: 1 and 8, SEQ ID NOs: 2 and 9, SEQ ID NOs: 3 and 10, SEQ ID NOs: 4 and 11, SEQ ID NOs: 5 and 12, SEQ ID NOs: 6 and 13, SEQ ID NOs: 7 and 14.

[0011] In some embodiments, the primer set comprises one or more pairs of GLA-F1 / R1 primer pairs, GLA-F2 / R2 primer pairs, GLA-F3 / R3 primer pairs, and GLA-F4 / R4 primer pairs. In some preferred embodiments, the primer set comprises a GLA-F1 / R1 primer pair, a GLA-F2 / R2 primer pair, a GLA-F3 / R3 primer pair, or a GLA-F4 / R4 primer pair.

[0012] In some embodiments, the Fabry disease multiple mutations comprise GLA Point mutations in genes and GLA Insertions and deletions within genes; preferably, the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes.

[0013] In some preferred embodiments, the primer set further comprises GLA-Gap primers; wherein the GLA-Gap primers comprise primers whose sequences are shown in SEQ ID NOs: 15-54.

[0014] In some embodiments, the plurality of Fabry disease mutations further comprises a large deletion within the range of the GLA-Gap primer.

[0015] In the present invention, the GLA-F / R primer pair consists of a GLA-F primer and a GLA-R primer. For example, the GLA-F1 / R1 primer pair consists of a GLA-F1 primer (SEQ ID NO: 1) and a GLA-R1 primer (SEQ ID NO: 8), the GLA-F2 / R2 primer pair consists of a GLA-F2 primer (SEQ ID NO: 2) and a GLA-R2 primer (SEQ ID NO: 9), the GLA-F3 / R3 primer pair consists of a GLA-F3 primer (SEQ ID NO: 3) and a GLA-R3 primer (SEQ ID NO: 10), the GLA-F4 / R4 primer pair consists of a GLA-F4 primer (SEQ ID NO: 4) and a GLA-R4 primer (SEQ ID NO: 11), and the GLA-F5 / R5 primer pair consists of a GLA-F5 primer (SEQ ID NO: 5) and a GLA-R5 primer (SEQ ID NO: 6). 12), the GLA-F6 / R6 primer pair consists of a GLA-F6 primer (SEQ ID NO: 6) and a GLA-R6 primer (SEQ ID NO: 13), and the GLA-F7 / R7 primer pair consists of a GLA-F7 primer (SEQ ID NO: 7) and a GLA-R7 primer (SEQ ID NO: 14). Any pair or combination of the above primer pairs can be used to achieve the technical effects of the present invention. The GLA-F primer and the GLA-R primer according to the present invention can amplify the GLA-F and GLA-R primers on the genome. GLA The complete gene sequence includes any type of mutation sequence within the primer range. The GLA-Gap primers according to the present invention can amplify the deletion fragment within the GLA-Gap primer range and detect unknown large-fragment deletion type mutations.

[0016] In some specific embodiments, the primer set comprises one or more pairs of GLA-F / R primer pairs and all GLA-Gap primers; preferably, the primer set comprises one or more pairs of GLA-F1 / R1 primer pairs, GLA-F2 / R2 primer pairs, GLA-F3 / R3 primer pairs, GLA-F4 / R4 primer pairs and all GLA-Gap primers; more preferably, the primer set comprises GLA-F1 / R1 primer pairs and all GLA-Gap primers. The above primer sequences are shown in Table 1, and the primer positions are shown in Table 1. Figure 1 shown.

[0017] In some embodiments, the length of the amplified product of the GLA-F1 / R1 primer pair, the GLA-F2 / R2 primer pair, the GLA-F3 / R3 primer pair, the GLA-F4 / R4 primer pair, the GLA-F5 / R5 primer pair, the GLA-F6 / R6 primer pair, and the GLA-F7 / R7 primer pair is about 11.5 kb. Preferably, if there is a SNP at the primer position, a degenerate base primer is used.

[0018] In some embodiments, the GLA-Gap primers are within a range of about 300-400 kb. The GLA-Gap primers can detect large deletions within a range of about 300-400 kb.

[0019] In some embodiments, the 5' end of the primer of the primer set further comprises a DNA barcode, and the DNA barcode is used to distinguish different samples. In some preferred embodiments, the 5' end barcodes of the GLA-F primer and the GLA-R primer can be the same or different, and those skilled in the art can select as needed. In some embodiments, the length of the DNA barcode is 5-50nt.

[0020] In some embodiments, the primer set is used to simultaneously amplify multiple Fabry disease mutations in one reaction system. In some preferred embodiments, the primer set is used to amplify a complete Fabry disease mutation in one reaction system by long-range PCR. GLA Gene fragments and unknown large missing fragments.

[0021] The primer set according to the present invention can be used for PCR amplification of a reaction system including Fabry disease-related pathogenic range GLA Fragments of various mutation types of genes. Combined with subsequent PacBio or Nanopore sequencing platforms, it can detect GLA All mutation types of gene segments.

[0022] The second aspect of the present invention provides the use of the primer set in preparing a kit for detecting multiple mutations of Fabry disease.

[0023] The third aspect of the present invention provides a kit for simultaneously detecting multiple mutations of Fabry disease, which comprises the following reagents: (1) a reagent for long-fragment PCR amplification; (2) a reagent for constructing a third-generation sequencing library; wherein the reagent for long-fragment PCR amplification comprises the primer set described in the present invention.

[0024] In some embodiments, the Fabry disease multiple mutations comprise GLA Point mutations in genes, GLAIntragenic insertions and deletions and / or large deletions within the GLA-Gap primer range; preferably, the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes.

[0025] In some embodiments, the reagents for long-fragment PCR amplification further include DNA polymerase and reaction buffer. In some embodiments, the PCR amplification product may be purified or not before the next step of reaction, and those skilled in the art may choose as needed.

[0026] In some embodiments, the reagents for constructing a third-generation sequencing library include adapters, ligases, DNA purification magnetic beads, reaction buffers, and exonucleases.

[0027] In some embodiments, the reagents for long-fragment PCR amplification are used in one reaction system. For example, the PCR amplification is completed in one reaction tube.

[0028] In some embodiments, the third generation sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0029] In a specific embodiment, PacBio library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0030] In a specific embodiment, the PacBio universal blunt-end adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 55), which is annealed to form a blunt-end stem-loop structure adapter adapter. DNA (barcode) with different sequences of 5-50nt can be added to the stem to form adapter adapters with different barcodes. PacBio libraries with different barcodes can be mixed together for sequencing.

[0031] In a specific embodiment, the PacBio universal TA adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 56), and a blunt-ended stem-loop structure adapter adapter is formed by annealing. DNA (barcode) of different sequences of 5-50nt can be added to the stem to form adapter adapters with different barcodes. PacBio libraries with different barcodes can be mixed together for sequencing.

[0032] In some embodiments, the PacBio connector may or may not have a barcode. Preferably, the PacBio connector has a barcode designed by PacBio or a self-designed barcode, and those skilled in the art can select according to their needs.

[0033] In some preferred embodiments, the PacBio library is compatible with the Pacific Biosciences sequencing platform.

[0034] In some preferred embodiments, the reagents used to construct the third-generation Nanopore library include end repair enzymes, linkers, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.

[0035] In some embodiments, Nanopore library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0036] In some embodiments, the Nanopore connector may or may not have a barcode. Preferably, the Nanopore connector has a barcode designed by ONT or a self-designed barcode, and those skilled in the art can select one according to their needs.

[0037] In some preferred embodiments, the Nanopore library matches the ONT sequencing platform.

[0038] The fourth aspect of the present invention provides a system for simultaneously detecting multiple mutations of Fabry disease, which comprises the following modules: (1) an acquisition module: used to obtain and prepare a sample from a subject; (2) an amplification module: used to perform long-fragment PCR amplification on the sample; (3) a library construction module: used to construct a third-generation sequencing library; (4) a sequencing module: used to sequence and analyze mutations; wherein the long-fragment PCR amplification in the acquisition module is performed using the primer set described in the present invention or the kit described in the present invention.

[0039] In some embodiments, the Fabry disease multiple mutations comprise GLA Point mutations in genes, GLA Intragenic insertions and deletions and / or large deletions within the GLA-Gap primer range; preferably, the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes.

[0040] In some embodiments, the sample is selected from a biological sample or gDNA extracted from the biological sample; preferably, 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.

[0041] In some embodiments, the long-fragment PCR amplification in the acquisition module is performed in one reaction system.

[0042] In some embodiments, the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0043] The fifth aspect of the present invention provides a method for simultaneously detecting multiple mutations of Fabry disease, comprising: (1) obtaining and preparing a sample from a subject; (2) performing long-fragment PCR amplification on the sample; (3) constructing a third-generation sequencing library; (4) sequencing and analyzing mutations; wherein the long-fragment PCR amplification is performed using the primer set described in the present invention or the kit described in the present invention.

[0044] The sixth aspect of the present invention provides a method for detecting the cis-trans forms of multiple mutations of Fabry disease, comprising: (1) obtaining and preparing a sample from a subject; (2) performing long-fragment PCR amplification on the sample; (3) constructing a third-generation sequencing library; (4) sequencing and analyzing the cis-trans forms of the mutations; wherein the long-fragment PCR amplification is performed using the primer set described in the present invention or the kit described in the present invention.

[0045] In some embodiments, the Fabry disease multiple mutations comprise GLA Point mutations in genes, GLA Intragenic insertions and deletions and / or large deletions within the GLA-Gap primer range; preferably, the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes.

[0046] The cis-trans configuration of a mutation refers to the relative relationship of the mutation positions in the genotype. A cis mutation is when two or more mutations are located on the same chromosome, i.e., they are on the same allele. In the cis case, if a gene has two mutations, both mutations are passed on to offspring together. The cis configuration may affect the function of the gene in some cases, for example, if both mutations are deleterious, they may have a more severe effect than either mutation alone. A trans mutation is when two or more mutations are located on different chromosomes, i.e., they are on different alleles. In the trans case, if an individual inherits two mutations from one parent, the two mutations are located on two different chromosomes. The trans configuration usually means that the mutations will not be passed on to offspring together unless chromosome exchange occurs (such as crossing over during meiosis). Therefore, detecting mutation cis-trans configuration is very important for understanding inheritance patterns and predicting the risk of genetic diseases. For example, for a recessive single gene disease, an individual with a cis mutation is a carrier. Since the two or more mutations are located on the same allele, they are not pathogenic, and these two or more mutations will not necessarily be passed on to offspring. However, an individual with a trans mutation is a patient with the recessive single gene disease. Since the two or more mutations are located on different alleles, they are pathogenic, and one of the two or more mutations will inevitably be passed on to offspring.

[0047] In some embodiments, the sample is selected from a biological sample or gDNA extracted from the biological sample; preferably, 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.

[0048] In some embodiments, the long-fragment PCR amplification is performed in one reaction system.

[0049] In some embodiments, the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0050] The present invention based on the specific combination of long-fragment PCR amplification and third-generation high-throughput sequencing can detect FD pathogenic genes in multiple samples simultaneously with high specificity, accuracy and rapidity. GLA Various related mutations.

[0051] The excellent technical effects of the method and kit of the present invention are mainly in the following aspects: (1) Wide detection range. The present invention can simultaneously detect all the FDs that have been discovered. GLA Point mutations, insertions and deletions of genes, as well as large deletions of about 400kb within the range of GLA-Gap primers.

[0052] (2) Detection of multiple mutation types in a single system. Traditional methods require a detection system to be set up for each mutation type, while the present invention detects multiple mutations simultaneously in one reaction primer system, including GLA Point mutations, insertions and deletions, and large deletions of genes.

[0053] (3) Low false positive and missed detection rates. Currently, the most common method for detecting FD is biochemical testing, while the first-generation sequencing in the molecular diagnosis of FD cannot be applied on a large scale and has a small detection range; the second-generation sequencing process for FD detection is more complicated, and the judgment of the mutation structure type (mutation cis-trans, gene insertion and deletion information, etc.) is not very clear; MLPA can only detect known point mutations and large fragment deletions, and the process is complicated and cannot be applied on a large scale; the present invention can detect the above mutation types at the same time, and the judgment is more accurate and comprehensive.

[0054] (4) Sample diversification. The template used for PCR can be peripheral blood, dried blood spots or extracted genomic DNA, or human cell lines or other specific tissues.

[0055] (5) High-throughput detection. The third-generation sequencing can realize 384 kinds of barcode adapters. In fact, more barcode adapters can be designed according to needs. Or a dual barcode system with primers and adapters can be used to achieve more barcode combinations. The high-throughput characteristics of the third-generation sequencing platform determine that high-throughput sample detection can be achieved.

[0056] (6) High accuracy. PacBio's dumbbell-shaped library can be read multiple times during sequencing, and the base accuracy of the sequencing results after correction is greater than 99%. In addition, PacBio sequencing errors are random, and the base accuracy is greater than 99.9% after correction through sequencing depth. Therefore, gene mutations within the primer detection range can be accurately read.

[0057] (7) Flexible detection time. The Nanopore platform can generate data within minutes, and data analysis can be started within minutes or hours depending on the actual data volume. When the detection timeliness requirements are relatively high, the Nanopore platform has a time advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Fabry disease GLA Schematic diagram of primer design for gene detection.

[0059] Figure 2 Fabry disease GLA Schematic diagram of gel electrophoresis of gene amplified fragments.

[0060] Figure 3-6Fabry disease GLA PacBio sequencing results of gene mutation samples, including Figure 3 yes GLA Gene exon point mutation samples, Figure 4 yes GLA Gene intron point mutation samples, Figure 5 yes GLA Gene double intron mutation samples, Figure 6 yes GLA Gene intron insertion samples. DETAILED DESCRIPTION

[0061] Exemplary embodiments of the present invention will now be described in detail, which should not be considered as limiting the present invention but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0062] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope. Although any methods and materials similar or equivalent to those described herein can be used in the practice or test of the present disclosure, what is described below is preferred materials and methods.

[0063] The sequences used in the present invention

[0064]

[0065] Example 1: Using the long-fragment PCR method of the present invention to amplify FD-related genes using different primer combinations GLA fragment; wherein the primer combination includes one of the following primer pairs (and their corresponding sequences): GLA-F1 / R1 (SEQ ID NOs: 1 and 8), GLA-F2 / R2 (SEQ ID NOs: 2 and 9), GLA-F3 / R3 (SEQ ID NOs: 3 and 10), GLA-F4 / R4 (SEQ ID NOs: 4 and 11), GLA-F5 / R5 (SEQ ID NOs: 5 and 12), GLA-F6 / R6 (SEQ ID NOs: 6 and 13), and GLA-F7 / R7 (SEQ ID NOs: 7 and 14).

[0066] Prepare the reaction system according to Table 2 (Long-range PCR) below to amplify peripheral blood, dried blood spots, and genomic DNA samples:

[0067] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 3 below:

[0068] After amplification, 5 μL of each sample was taken and tested on a 1% DNA gel. The results were as follows: Figure 2 As shown, different GLA primer combinations were screened, and a few GLA primer combinations could be effectively amplified, and primer combinations GLA-F1 / R1, GLA-F2 / R2, GLA-F3 / R3, and GLA-F4 / R4 were effectively amplified.

[0069] Example 2: Construction of a PacBio sequencing library using the PCR method of the present invention Step 1: Long-range PCR amplification According to Table 4 (long fragment PCR) below, the reaction system was prepared, and the GLA-F1, GLA-R1 and GLA-Gap effectively amplified in Example 1 were used to amplify FD-related genes of different mutation types. GLA Fragmented peripheral blood samples.

[0070]

[0071] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 5 below:

[0072] After amplification, the amplified product was placed in a centrifuge at 10,000 rpm for 20 minutes. After centrifugation, the product was placed horizontally and 4 μL of the supernatant was added to a new tube.

[0073] Step 2: Construction of PacBio sequencing library Prepare the reaction system according to Table 6 below:

[0074]

[0075] The reaction was carried out in a PCR machine under the following conditions: 37ºC 20 min; 25ºC 15 min; 65ºC 10 min. After the reaction was completed, 0.5 μL Exonuclease III (NEB, Cat#M0206L) and 0.5 μL Exonuclease VII (NEB, Cat#M0379L) were added and the reaction was continued at 37ºC for 1 hour. The DNA was purified twice using 0.6x Ampure PB magnetic beads (PacBio, Cat#100-265-900) according to the manufacturer's instructions, and finally eluted with 10μL Elution Buffer. The obtained DNA eluate is the target DNA PacBio sequencing library. The DNA concentration was measured on the Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216) using Qubit dsDNA HS reagent (ThermoFisher, Cat# Q32851). When there are multiple sample PacBio sequencing libraries, equal amounts of the libraries can be mixed together to prepare a mixed library.

[0076] Step 3: PacBio sequencing and analysis According to the total concentration and molar concentration of the library, an appropriate volume of the library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primer (PacBio, Cat#100-970-100) to prepare the final library that can be used in the machine. Representative sequencing results are shown in Figure 3-6 As shown, Figure 3 for GLA Gene exon point mutation samples, Figure 4 for GLA Gene intron point mutation samples, Figure 5 for GLA Gene double intron mutation samples, Figure 6 for GLA Gene intron insertion samples.

[0077] Embodiment 3: GLA Detection and verification of gene mutations The peripheral blood genomic DNA of 73 subjects was collected as validation samples, a total of 73 cases, and the method (and kit) of the present invention was used to simultaneously detect GLA Multiple mutations of genes. At the same time, the method of GLA exon Sanger sequencing was used to detect point mutations and deletions of related genes. The results obtained by the present invention were compared with the control results. The results are shown in Table 7, among which the results of 69 samples were completely consistent, and the method of the present invention detected mutations in 4 more samples compared with the method of GLA exon Sanger sequencing, and the verification was correct.

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Therefore, the results detected by the method of the present invention, after comparison with the GLA exon Sanger sequencing method, both the specificity and the sensitivity reached 100%.

[0084] It should be noted that although some features of the present invention have been illustrated by the above embodiments, they cannot be used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. The reaction reagents, reaction conditions, etc. involved in the long-fragment PCR reaction and the construction of the third-generation sequencing library can be adjusted and changed accordingly according to specific needs. Therefore, for those skilled in the art, several simple substitutions can be made without departing from the concept and principle of the present invention, which should all be included in the protection scope of the present invention.

Claims

1. A primer set for simultaneously amplifying multiple mutations of Fabry disease, wherein the primer set comprises one or more pairs of GLA-F1 / R1 primer pair, GLA-F2 / R2 primer pair, GLA-F3 / R3 primer pair, GLA-F4 / R4 primer pair, GLA-F5 / R5 primer pair, GLA-F6 / R6 primer pair, and GLA-F7 / R7 primer pair; wherein the nucleotide sequences of the GLA-F1 / R1 primer pair, GLA-F2 / R2 primer pair, GLA-F3 / R3 primer pair, GLA-F4 / R4 primer pair, GLA-F5 / R5 primer pair, GLA-F6 / R6 primer pair, and GLA-F7 / R7 primer pair are SEQ ID NOs: 1 and 8, SEQ ID NOs: 2 and 9, SEQ ID NOs: 3 and 10, SEQ ID NOs: 4 and 11, SEQ ID NOs: 5 and 12, SEQ ID NOs: 6 and 13, SEQ ID NOs: 7 and 14. 2 . The primer set according to claim 1 , wherein the primer set comprises one or more pairs of a GLA-F1 / R1 primer pair, a GLA-F2 / R2 primer pair, a GLA-F3 / R3 primer pair, or a GLA-F4 / R4 primer pair.

3. The primer set according to claim 1, wherein the multiple Fabry disease mutations comprise GLA Point mutations in genes and GLA Intragenic insertions and deletions.

4. The primer set according to claim 3, wherein the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes.

5. The primer set according to claim 1, wherein the primer set further comprises a GLA-Gap primer; wherein the GLA-Gap primer comprises a primer whose sequence is shown in SEQ ID NO: 15-54.

6. The primer set according to claim 5, wherein the multiple Fabry disease mutations further comprise a large deletion within the range of the GLA-Gap primer.

7. The primer set according to claim 1, wherein the length of the amplification product of the GLA-F1 / R1 primer pair, GLA-F2 / R2 primer pair, GLA-F3 / R3 primer pair, GLA-F4 / R4 primer pair, GLA-F5 / R5 primer pair, GLA-F6 / R6 primer pair, and GLA-F7 / R7 primer pair is 11.5 kb.

8. The primer set according to claim 5, wherein the GLA-Gap primer ranges from 300 to 400 kb. 9 . The primer set according to claim 1 or 5 , wherein the 5′ end of the primer in the primer set further comprises a DNA barcode. 10 . The primer set according to claim 9 , wherein the DNA barcodes are the same or different, and the length of the DNA barcodes is 5-50 nt.

11. The primer set according to claim 1 or 5, wherein the primer set simultaneously amplifies multiple Fabry disease mutations in one reaction system.

12. Use of the primer set according to any one of claims 1 to 10 in preparing a kit for detecting multiple mutations of Fabry disease.

13. A kit for simultaneously detecting multiple mutations of Fabry disease, comprising the following reagents: (1) Reagents for long-range PCR amplification; (2) Reagents for constructing third-generation sequencing libraries; in, The reagents for long-fragment PCR amplification comprise the primer set according to any one of claims 1 to 11.

14. The kit according to claim 13, wherein the multiple Fabry disease mutations comprise GLA Point mutations in genes, GLA Intragenic insertions and deletions and / or large deletions within the range of the GLA-Gap primers.

15. The kit according to claim 14, wherein the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes. The kit according to claim 13 , wherein the reagents for long-fragment PCR amplification further comprise a DNA polymerase and a reaction buffer.

17. The kit according to claim 13, wherein the reagents for constructing a third-generation sequencing library comprise adapters, ligases, DNA purification magnetic beads, reaction buffers and exonucleases.

18. The kit according to claim 13, wherein the reagents for long-fragment PCR amplification are used in one reaction system.

19. The kit according to claim 13, wherein the third generation sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

20. A system for simultaneously detecting multiple mutations of Fabry disease, comprising the following modules: (1) Collection module: used to obtain and prepare subject samples; (2) Amplification module: used to perform long-fragment PCR amplification on the sample; (3) Library construction module: used to construct the third-generation sequencing library; (4) Sequencing module: used for sequencing and analyzing mutations; The long-fragment PCR amplification in the acquisition module is performed using a primer set as described in any one of claims 1 to 11 or a kit as described in any one of claims 12 to 19.

21. The system of claim 20, wherein the plurality of Fabry disease mutations comprises GLA Point mutations in genes, GLA Intragenic insertions and deletions and / or large deletions within the range of the GLA-Gap primers.

22. The system of claim 21, wherein the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes.

23. The system of claim 20, wherein the sample is selected from a biological sample or gDNA extracted from the biological sample.

24. The system of claim 23, 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.

25. The system according to claim 20, wherein the long-fragment PCR amplification in the acquisition module is performed in one reaction system.

26. The system of claim 20, wherein the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

27. A method for detecting cis-trans forms of multiple mutations in Fabry disease, comprising: (1) Obtaining and preparing subject samples; (2) performing long-fragment PCR amplification on the sample; (3) Construction of third-generation sequencing library; (4) sequencing and analyzing the cis and trans forms of the mutation; The long-fragment PCR amplification is performed using the primer set described in any one of claims 1 to 11 or the kit described in any one of claims 12 to 19.

28. The method of claim 27, wherein the plurality of Fabry disease mutations comprises GLA Point mutations in genes, GLA Intragenic insertions and deletions and / or large deletions within the range of the GLA-Gap primers.

29. The method according to claim 28, wherein the GLA Intragenic insertions and deletions include GLA Insertions and deletions in the exon regions of genes and GLA Insertions and deletions in the intronic regions of genes.

30. The method of claim 27, wherein the sample is selected from a biological sample or gDNA extracted from the biological sample.

31. The method of claim 30, 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.

32. The method according to claim 27, wherein the long-fragment PCR amplification is performed in one reaction system.

33. The method according to claim 27, wherein the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

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