Primer set, kit and application thereof for detecting MUC1 gene mutation in autosomal dominant tubulointerstitial nephropathy

Abstract

The present invention relates to a primer set, a kit and an application thereof for detecting a mutation in the MUC1 gene for autosomal dominant tubulointerstitial nephropathy. The present invention provides a primer set comprising one or more pairs of primer pairs selected from the group consisting of a MUC1-F1 / R1 primer pair, a MUC1-F2 / R2 primer pair, a MUC1-F3 / R3 primer pair, a MUC1-F4 / R4 primer pair, a MUC1-F5 / R5 primer pair, a MUC1-F6 / R6 primer pair and a MUC1-F7 / R7 primer pair; and further comprising a MUC1-Gap primer. The present invention based on a specific combination of long-fragment PCR amplification and third-generation high-throughput sequencing can detect ADTKD pathogenic genes in multiple samples simultaneously with high specificity, accuracy and speed. MUC1 Various related mutations.

Description

Technical Field

[0001] The present invention relates to autosomal dominant tubulointerstitial kidney disease (ADTKD; Autosomal Dominant TubμLo interstitial Kidney Disease) MUC1 The invention relates to a primer set and a kit for detecting multiple mutations of a gene and their application, specifically to a primer and a method for detecting multiple mutations of ADTKD-MUC1 using a third-generation long-read sequencing platform, and a kit suitable for this method. Background Art

[0002] Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a group of inherited kidney diseases characterized by tubular damage and interstitial fibrosis in the absence of glomerular lesions, which inevitably progress to end-stage renal disease. The epidemiology of ADTKD cannot be precisely determined because of the rarity of these diseases, recent clinical features, and high underdiagnosis. There is no evidence that the prevalence of the disease varies significantly between different countries, regions, and ethnic populations, and there are no reports of sex differences in the prevalence or clinical manifestations of ADTKD.

[0003] ADTKD is caused by mutations in at least five different genes: UMOD, MUC1, HNF1b, REN, and SEC61A1. UMOD and MUC1 mutations are the most common subtypes of ADTKD. Patients with ADTKD-MUC1 develop ESRD at different rates, with dominant inheritance and slowly progressive chronic kidney disease as the main clinical features, without other typical clinical manifestations.

[0004] The MUC1 gene encodes mucin-1, a transmembrane glycoprotein widely distributed in the distal renal tubules, which plays an important role in maintaining the tubular lumen. MUC1 has a variable number of tandem repeats (VNTR) domain, and a cytosine deoxynucleotide is inserted into this domain, causing a frameshift mutation in the gene, resulting in a new peptide chain that cannot fold into a protein with normal function and accumulates in the tubular epithelial cells, ultimately leading to tubular dysfunction and necrosis. However, its specific mechanism remains to be clarified. Six frameshift mutations that cause ADTKD-MUC1 have been reported, each of which results in abnormal MUC1fs protein.

[0005] Due to the complexity of the gene structure and high guanosine-cytosine content, conventional Sanger sequencing or NGS cannot routinely identify MUC1 mutations. Due to the size of the VNTR ranging from 1.5 kb to 7.5 kb and the sequence containing >80% GC, it is difficult to amplify by long-distance PCR, making sequence analysis more complicated. In addition, cytosine repeats are family-specific and occur in different units of repeats in each family. Currently, the ADTKD-MUC1 test only identifies cytosine repeats: there are other mutations that cause the same frameshift mutation, which cannot be easily sequenced at present. MUC1 has strong familial heterogeneity, so clinical development is difficult, but there are reports of using mass spectrometry, immunohistochemistry, targeted gene mutation analysis, and family genetic linkage analysis to find mutation sites in MUC1.

[0006] Based on the current detection method, it can be achieved MUC1 Detection of gene point mutations, but the detection has the following limitations:

[0007] 1. It is impossible to detect all mutation types simultaneously in the same system;

[0008] 2. Mass spectrometry relies on the detection of known specific mutation sites and is limited by existing spectral libraries. It has poor detection sensitivity and requires a large amount of starting DNA.

[0009] 3. Immunohistochemistry is difficult to perform renal biopsy, so it is not easy to obtain samples;

[0010] 4. Targeted gene mutation analysis can only detect point mutations within the VNTR region;

[0011] 5. First-generation sequencing and second-generation sequencing can only detect point mutations outside the VNTR region, and cannot detect mutations within the VNTR region. Summary of the invention

[0012] In view of this, the present invention provides a method based on long-fragment PCR amplification and third-generation sequencing to detect multiple mutations associated with ADTKD-MUC1. Long-fragment PCR amplification is achieved in one reaction tube and is used to detect MUC1 Point mutations in genes, MUC1 Insertions and deletions within genes, large deletions within the MUC1-Gap primer range. Combined with the read length measurement characteristics of the third-generation sequencing platform, accurate, rapid and high-throughput detection of ADTKD-MUC1 related gene mutations can be achieved. The method involved in the present invention is simple to operate, and the long-fragment PC and third-generation library are of reliable quality and strong repeatability, which is conducive to the application of third-generation sequencing technology in clinical detection.

[0013] The purpose of the present invention is to solve the problem of ADTKD pathogenic gene at present MUC1Incomplete detection coverage, missed detection by specific NGS detection process, and inability of NGS detection to distinguish between cis and trans structures of mutations. Amplification of ADTKD-related pathogenic genes by long-fragment PCR MUC1 The region and preparation of third-generation sequencing libraries can achieve the goal of comprehensive, accurate and rapid detection of multiple ADTKD mutations in multiple samples.

[0014] definition

[0015] Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is a group of genetic diseases characterized by the appearance of fluid-filled sacs (cysts) deep inside the kidneys, which eventually lead to chronic kidney disease with renal failure. The disease is caused by inherited gene defects. Currently, the known pathogenic genes associated with ADTKD include UMOD, REN, HNF1B and MUC1. Mutations in these genes may lead to abnormal development and dysfunction of the kidneys.

[0016] The protein encoded by the MUC1 gene is called Mucin 1, which is a transmembrane glycoprotein that belongs to the mucin family. Mutations in the MUC1 gene are associated with autosomal dominant tubulointerstitial nephropathy (ADTKD). This inherited disease is characterized by the development of cysts inside the kidneys, which may eventually lead to chronic kidney disease and renal failure. Misfolded proteins caused by mutations in the MUC1 gene may interfere with the normal function of cells, leading to damage to kidney tissue and the development of disease.

[0017] As used herein, the term "ADTKD-MUC1" refers to a gene that causes ADTKD. MUC1 ADTKD-MUC1 multiple mutations refers to a gene that is associated with autosomal dominant tubulointerstitial nephropathy. MUC1 Multiple mutations in genes associated with autosomal dominant tubulointerstitial nephropathy, including MUC1 Point mutations in genes, MUC1 Intragenic insertions and deletions and large deletions within the range of the MUC1-Gap primers.

[0018] Variable Number of Tandem Repeats (VNTR) is a special DNA sequence, also known as minisatellite DNA. VNTR consists of a series of short nucleotide sequences that are repeated in the genome, but the number of repetitions varies between different individuals. The length of VNTR is usually between 10 and several hundred nucleotides, and the number of repetitions can range from 10 to 1000 times.

[0019] As used herein, the term "VNTR region" refers to MUC1 VNTR region of the gene. In the MUC1 gene, mutations in the VNTR region usually cause autosomal dominant tubulointerstitial nephropathy (ADTKD-MUC1). Mutations in the VNTR region of the MUC1 gene are usually due to the insertion of an extra cytosine in the seven cytosine (C) sequence in the VNTR region, resulting in a frameshift mutation and producing an abnormal MUC1 protein (MUC1fs). The accumulation of this abnormal protein in renal tubular epithelial cells may be an important pathogenic mechanism of ADTKD-MUC1.

[0020] In the present invention, the cis-trans of a mutation refers to the relative relationship of the mutation position in a genotype. A cis mutation refers to two or more mutations located on the same chromosome, i.e., they are located on the same allele. In the cis case, if a gene has two mutations, the two mutations will be passed on to the offspring together. The cis configuration may affect the function of the gene in some cases, for example, if both mutations are harmful, they may produce a more serious effect than a single mutation. A trans mutation refers to two or more mutations located on different chromosomes, i.e., they are located on different alleles. In the trans case, if an individual inherits two mutations from his parents, the two mutations are located on two different chromosomes. The trans configuration usually means that the mutations will not be passed on to the offspring together unless chromosome exchange occurs (such as crossover exchange during meiosis). Therefore, detecting mutation cis-trans is very important for understanding inheritance patterns and predicting the risk of genetic diseases.

[0021] In a first aspect, the present invention provides a primer set for simultaneously amplifying multiple mutations of ADTKD-MUC1, wherein the primer set comprises one or more pairs of MUC1-F1 / R1 primer pairs, MUC1-F2 / R2 primer pairs, MUC1-F3 / R3 primer pairs, MUC1-F4 / R4 primer pairs, MUC1-F5 / R5 primer pairs, MUC1-F6 / R6 primer pairs and MUC1-F7 / R7 primer pairs; wherein the nucleotide sequences of the MUC1-F1 / R1 primer pair, the MUC1-F2 / R2 primer pair, the MUC1-F3 / R3 primer pair, the MUC1-F4 / R4 primer pair, the MUC1-F5 / R5 primer pair, the MUC1-F6 / R6 primer pair and the MUC1-F7 / R7 primer pair are as shown in 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, and SEQ ID NOs: 7 and 14.

[0022] In some embodiments, the primer set comprises one or more of a MUC1-F1 / R1 primer pair, a MUC1-F4 / R4 primer pair, a MUC1-F5 / R5 primer pair, and a MUC1-F7 / R7 primer pair.

[0023] In some preferred embodiments, the primer set comprises a MUC1-F1 / R1 primer pair, a MUC1-F4 / R4 primer pair, a MUC1-F5 / R5 primer pair, or a MUC1-F7 / R7 primer pair.

[0024] In some embodiments, the ADTKD-MUC1 multiple mutations include MUC1 Point mutations in genes and MUC1 Insertions and deletions within genes; preferably, the MUC1 The point mutations of the gene include point mutations within the VNTR region and point mutations outside the VNTR region.

[0025] In some preferred embodiments, the primer set further comprises MUC1-Gap primers; wherein the MUC1-Gap primers comprise primers whose sequences are shown in SEQ ID NOs: 15-52.

[0026] In some embodiments, the ADTKD-MUC1 multiple mutations further comprise a large deletion within the range of the MUC1-Gap primer.

[0027] In the present invention, the MUC1-F / R primer pair consists of a MUC1-F primer and a MUC1-R primer. For example, the MUC1-F1 / R1 primer pair consists of a MUC1-F1 primer (SEQ ID NO: 1) and a MUC1-R1 primer (SEQ ID NO: 8), the MUC1-F2 / R2 primer pair consists of a MUC1-F2 primer (SEQ ID NO: 2) and a MUC1-R2 primer (SEQ ID NO: 9), the MUC1-F3 / R3 primer pair consists of a MUC1-F3 primer (SEQ ID NO: 3) and a MUC1-R3 primer (SEQ ID NO: 10), the MUC1-F4 / R4 primer pair consists of a MUC1-F4 primer (SEQ ID NO: 4) and a MUC1-R4 primer (SEQ ID NO: 11), and the MUC1-F5 / R5 primer pair consists of a MUC1-F5 primer (SEQ ID NO: 5) and a MUC1-R5 primer (SEQ ID NO: 6). 12), the MUC1-F6 / R6 primer pair consists of a MUC1-F6 primer (SEQ ID NO: 6) and a MUC1-R6 primer (SEQ ID NO: 13), and the MUC1-F7 / R7 primer pair consists of a MUC1-F7 primer (SEQ ID NO: 7) and a MUC1-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.

[0028] The MUC1-F primer and the MUC1-R primer according to the present invention can amplify the genome within the range of the MUC1-F and MUC1-R primers. MUC1 The complete gene sequence includes any type of mutation sequence within the primer range. The MUC1-Gap primer according to the present invention can amplify the deletion fragment within the MUC1-Gap primer range and detect unknown large-fragment deletion type mutations.

[0029] In some specific embodiments, the primer set comprises one or more pairs of MUC1-F / R primer pairs and all MUC1-Gap primers; preferably, the primer set comprises one or more pairs of MUC1-F1 / R1 primer pairs, MUC1-F4 / R4 primer pairs, MUC1-F5 / R5 primer pairs, and MUC1-F7 / R7 primer pairs and all MUC1-Gap primers; more preferably, the primer set comprises MUC1-F1 / R1 primer pairs and all MUC1-Gap primers. The above primer sequences are shown in Table 1, and the primer positions are shown in Table 1. Figure 1 shown.

[0030] In some embodiments, the length of the amplified product of the MUC1-F1 / R1 primer pair, MUC1-F2 / R2 primer pair, MUC1-F3 / R3 primer pair, MUC1-F4 / R4 primer pair, MUC1-F5 / R5 primer pair, MUC1-F6 / R6 primer pair or MUC1-F7 / R7 primer pair is about 7.2 kb-7.8 kb, preferably 7.5 kb. Preferably, if there is a SNP at the primer position, a degenerate base primer is used.

[0031] In some embodiments, the MUCl-Gap primers have a range of about 400 kb. The MUCl-Gap primers can detect large deletions of about 400 kb within the MUCl-Gap primer range.

[0032] 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 MUC1-F primer and the MUC1-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 5-50 nt.

[0033] In some embodiments, the primer set is used to simultaneously amplify multiple mutations of ADTKD-MUC1 in one reaction system. In some preferred embodiments, the primer set is used to amplify a complete long-range PCR in one reaction system. MUC1 Gene fragments and unknown large missing fragments.

[0034] The primer set according to the present invention can be used for PCR amplification of a reaction system including ADTKD-related pathogenic range MUC1 Fragments of various mutation types of genes. Combined with subsequent PacBio or Nanopore sequencing platforms, it can detect MUC1 All mutation types of gene segments.

[0035] The second aspect of the present invention provides the use of the primer set in preparing a kit for detecting multiple mutations of ADTKD-MUC1.

[0036] The third aspect of the present invention provides a kit for simultaneously detecting multiple mutations of ADTKD-MUC1, 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.

[0037] In some embodiments, the ADTKD-MUC1 multiple mutations include MUC1Point mutations in genes, MUC1 Intragenic insertions and deletions and / or large deletions within the MUC1-Gap primer range; preferably, the MUC1 The point mutations of the gene include point mutations within the VNTR region and point mutations outside the VNTR region.

[0038] 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.

[0039] In some embodiments, the reagents for constructing a third-generation sequencing library include adapters, ligases, DNA purification magnetic beads, reaction buffers, and exonucleases.

[0040] 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.

[0041] In some embodiments, the third generation sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0042] In a specific embodiment, PacBio library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0043] In a specific embodiment, the PacBio universal blunt-end adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 53), 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.

[0044] In a specific embodiment, the PacBio universal TA adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 54), and a blunt-ended stem-loop structure adapter adapter is formed by annealing. 5-50nt of different sequence DNA (Barcode) can be added to the stem to form adapter adapters with different Barcodes. PacBio libraries with different Barcodes can be mixed together for sequencing.

[0045] 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.

[0046] In some preferred embodiments, the PacBio library is compatible with the Pacific Biosciences sequencing platform.

[0047] 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.

[0048] In some embodiments, Nanopore library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0049] 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.

[0050] In some preferred embodiments, the Nanopore library matches the ONT sequencing platform.

[0051] The fourth aspect of the present invention provides a system for simultaneously detecting multiple mutations of ADTKD-MUC1, 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.

[0052] In some embodiments, the ADTKD-MUC1 multiple mutations include MUC1 Point mutations in genes, MUC1 Intragenic insertions and deletions and / or large deletions within the MUC1-Gap primer range; preferably, the MUC1 The point mutations of the gene include point mutations within the VNTR region and point mutations outside the VNTR region.

[0053] 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.

[0054] In some embodiments, the long-fragment PCR amplification in the acquisition module is performed in one reaction system.

[0055] In some embodiments, the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0056] The fifth aspect of the present invention provides a method for simultaneously detecting multiple mutations of ADTKD-MUC1, comprising: (1) obtaining and preparing a subject sample; (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.

[0057] The sixth aspect of the present invention provides a method for detecting the cis-trans forms of multiple mutations in ADTKD-MUC1, 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.

[0058] In some embodiments, the ADTKD-MUC1 multiple mutations include MUC1 Point mutations in genes, MUC1 Intragenic insertions and deletions and / or large deletions within the MUC1-Gap primer range; preferably, the MUC1 The point mutations of the gene include point mutations within the VNTR region and point mutations outside the VNTR region.

[0059] 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.

[0060] In some embodiments, the long-fragment PCR amplification is performed in one reaction system.

[0061] In some embodiments, the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0062] The present invention based on the specific combination of long-fragment PCR amplification and third-generation high-throughput sequencing can realize the simultaneous detection of ADTKD pathogenic genes in multiple samples with high specificity, accuracy and rapidity. MUC1 Various related mutations.

[0063] The excellent technical effects of the method and kit of the present invention are mainly in the following aspects:

[0064] (1) Wide detection range. The present invention can simultaneously detect the ADTKD that has been discovered MUC1 Point mutations, insertions and deletions of the gene, as well as large deletions of approximately 400 Kb within the range of the MUC1-Gap primers.

[0065] (2) Detection of multiple mutation types with a single kit. 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 MUC1 Point mutations, insertions and deletions, and large deletions of genes.

[0066] (3) Low false positive and missed detection rates. Currently, the commonly used methods for detecting MUC1 are mass spectrometry and immunohistochemistry. In the molecular diagnosis of MUC1, first-generation sequencing and second-generation sequencing can only detect mutations outside the VNTR region, but cannot detect mutations within the VNTR region, and the judgment of the mutation structure type (mutation cis-trans, gene insertion and deletion information, etc.) is not clear. The present invention can detect the above mutation types at the same time, and the judgment is more accurate and comprehensive.

[0067] (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.

[0068] (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.

[0069] (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.

[0070] (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

[0071] Figure 1 Autosomal dominant tubulointerstitial nephropathy MUC1 Schematic diagram of primer design for gene detection.

[0072] Figure 2 Autosomal dominant tubulointerstitial nephropathy MUC1 Schematic diagram of gel electrophoresis of gene amplified fragments, showing the DNA gel electrophoresis diagram of different primer combinations amplified according to the PCR method in Example 1.

[0073] Figure 3-Figure 4 It is a representative autosomal dominant tubulointerstitial nephropathy MUC1 PacBio sequencing results of gene mutation samples. Figure 3 This is a sample of MUC1 gene point mutation in autosomal dominant tubulointerstitial nephropathy; Figure 4 The sample reads for the MUC1 gene point mutation in autosomal dominant tubulointerstitial nephropathy are displayed. DETAILED DESCRIPTION

[0074] 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.

[0075] 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.

[0076] The sequences used in the present invention are shown in Table 1.

[0077] Table 1:

[0078]

[0079] Example 1: Using the long fragment PCR method of the present invention, the ADTKD-related genes were amplified using primer combinations of MUC1-F1 / R1 (SEQ ID NOs: 1 and 8), MUC1-F2 / R2 (SEQ ID NOs: 2 and 9), MUC1-F3 / R3 (SEQ ID NOs: 3 and 10), MUC1-F4 / R4 (SEQ ID NOs: 4 and 11), MUC1-F5 / R5 (SEQ ID NOs: 5 and 12), MUC1-F6 / R6 (SEQ ID NOs: 6 and 13), and MUC1-F7 / R7 (SEQ ID NOs: 7 and 14). MUC1 Fragment.

[0080] Prepare the reaction system according to Table 2 (Long-range PCR) below to amplify peripheral blood, dried blood spots, and genomic DNA samples:

[0081] Table 2:

[0082]

[0083] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 3 below:

[0084] Table 3:

[0085]

[0086] 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, the primer combinations of MUC1-F1 / R1, MUC1-F2 / R2, MUC1-F3 / R3, MUC1-F4 / R4, MUC1-F5 / R5, MUC1-F6 / R6, and MUC1-F7 / R7 were screened, among which the primer combinations of MUC1-F1 / R1, MUC1-F4 / R4, MUC1-F5 / R5, and MUC1-F7 / R7 could be effectively amplified.

[0087] Example 2: Construction of a PacBio sequencing library using the PCR method of the present invention

[0088] Prepare the reaction system according to Table 4 (long fragment PCR) below, and use the MUC1-F1, MUC1-R1 and MUC1-Gap effectively amplified in Example 1 to amplify ADTKD-related genes of different mutation types MUC1 Fragmented peripheral blood samples:

[0089] Table 4:

[0090]

[0091] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 5 below:

[0092] Table 5:

[0093]

[0094] 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 mL of supernatant was added to a new tube.

[0095] Prepare the reaction system according to Table 6 below:

[0096] Table 6:

[0097]

[0098] 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 mL Exonuclease III (NEB, Cat#M0206L) and 0.5 mL 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.

[0099] 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 and Figure 4 As shown, Figure 3 for MUC1 Gene point mutation samples, Figure 4 for MUC1 Sequencing reads of gene point mutation samples.

[0100] Embodiment 3: MUC1 Detection and verification of gene mutations

[0101] Peripheral blood genomic DNA of 10 subjects was collected as validation samples, a total of 10 cases, and the method (and kit) of the present invention was used to simultaneously detect MUC1 Multiple mutations of genes. Enzyme digestion-mass spectrometry was used to detect point mutations and deletions of related genes. The results obtained by the present invention were compared with the control results, and the results are shown in Table 7, among which the results of 10 samples were completely consistent and verified to be correct.

[0102] Table 7:

[0103]

[0104] Therefore, the results of the detection by the method of the present invention, after comparison with the MUC1 exon enzyme digestion-mass spectrometry, both the specificity and the sensitivity reached 100%.

[0105] 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 ADTKD-MUC1, wherein the primer set comprises a pair of primers selected from the group consisting of MUC1-F1 / R1 primer pair, MUC1-F4 / R4 primer pair, MUC1-F5 / R5 primer pair and MUC1-F7 / R7 primer pair, and a MUC1-Gap primer; wherein the nucleotide sequences of the MUC1-F1 / R1 primer pair, the MUC1-F4 / R4 primer pair, the MUC1-F5 / R5 primer pair and the MUC1-F7 / R7 primer pair are as shown in SEQ ID NOs: 1 and 8, SEQ ID NOs: 4 and 11, SEQ ID NOs: 5 and 12, and SEQ ID NOs: 7 and 14, respectively, and the MUC1-Gap primer comprises primers having sequences as shown in SEQ ID NOs: 15-52; wherein the primer set simultaneously amplifies multiple mutations of ADTKD-MUC1 in one reaction system; The ADTKD-MUC1 multiple mutations include MUC1 Point mutations in genes, MUC1 Intragenic insertions and deletions and large deletions within the range of the MUC1-Gap primers, MUC1 The point mutations of the gene include point mutations within the VNTR region and point mutations outside the VNTR region. 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 . Use of the primer set according to claim 1 in preparing a kit for detecting multiple mutations of ADTKD-MUC1.

5. A kit for simultaneously detecting multiple mutations of ADTKD-MUC1, comprising the following reagents: (1) Reagents for long-range PCR amplification; (2) Reagents for constructing third-generation sequencing libraries; in, The reagent for long-fragment PCR amplification comprises the primer set described in any one of claims 1 to 3; The reagents for long-fragment PCR amplification are used in one reaction system; The ADTKD-MUC1 mutations include MUC1 Point mutations in genes, MUC1 Intragenic insertions and deletions and large deletions within the range of the MUC1-Gap primers, MUC1 The point mutations of the gene include point mutations within the VNTR region and point mutations outside the VNTR region. The kit according to claim 5 , wherein the reagents for long-fragment PCR amplification further comprise a DNA polymerase and a reaction buffer.

7. The kit according to claim 5, wherein the reagents for constructing a third-generation sequencing library comprise adapters, ligases, DNA purification magnetic beads, reaction buffers and exonucleases.

8. The kit according to claim 5, wherein the third-generation sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

9. A system for simultaneously detecting multiple mutations of ADTKD-MUC1, 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 3 or a kit as described in any one of claims 5 to 7; The long-fragment PCR amplification in the acquisition module is performed in one reaction system; The ADTKD-MUC1 mutations include MUC1 Point mutations in genes, MUC1 Intragenic insertions and deletions and large deletions within the range of the MUC1-Gap primers, MUC1 The point mutations of the gene include point mutations within the VNTR region and point mutations outside the VNTR region.

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

11. The system of claim 10, 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.

12. The system according to claim 9, wherein the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

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