Primer combination, kit and system for NOTCH2NLC gene detection

Abstract

The invention discloses a primer combination, a kit and a system for NOTCH2NLC gene detection, the primer combination comprises the following primer combinations: a first group of long-range amplification primer combination, a second group of long-range amplification primer combination, a third group of long-range amplification primer combination and a fourth group of long-range amplification primer combination, the first group of long-range amplification primer combination comprises primers with nucleotide sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2; a second group of long-range amplification primer combination, wherein the second group of long-range amplification primer combination comprises primers with nucleotide sequences as shown in SEQ ID NO: 6 and 7; and a repeated amplification primer combination, the repeated amplification primer combination comprising primers with nucleotide sequences as shown in SEQ ID NO: 3, 4 and 5. The primer combination provided by the invention is obtained through specific screening, the kit and the system based on the primer combination can detect more mutation types, insertion interruption of SNV, GGA and the like, lower-proportion chimerism and higher GGC repetition number can be detected, the operation is simple, and the detection cost is low.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a primer combination, a kit and a system for detecting the NOTCH2NLC gene. Background Art

[0002] Neuronal intranuclear inclusion disease (NIID) is a chronic progressive neurodegenerative disease, characterized by eosinophilic transparent intranuclear inclusions in neurons and somatic cells. The intranuclear inclusions may be composed of abnormal proteins such as intranuclear polyglycine proteins and are related to cell damage. NIID has a wide range of clinical manifestations, such as dementia, ataxia, developmental delay, Parkinson's disease, tremors, neuropathy and autonomic dysfunction have been reported.

[0003] NOTCH2NLC is one of the three human-specific NOTCH2NL genes (NOTCH2NLA, NOTCH2NLB and NOTCH2NLC). The NOTCH2NL gene sequences have extremely high similarity and contain many GC-rich regions, making it very difficult to analyze and detect the NOTCH2NLC gene sequence.

[0004] There is a GGC repeat sequence in the 5'UTR region of the NOTCH2NLC gene. Studies have shown that the repeat amplification of this sequence is related to diseases such as neuronal intranuclear inclusion disease and essential tremor. In normal people, the (GGC) n repeat number of the NOTCH2NLC gene does not exceed 38 times, and the pathological mutation (GGC) n repeat number exceeds 66 times. Among them, the clinical manifestations of patients with a repeat number below 100 times are mainly Parkinson's syndrome; the clinical manifestations of patients with a repeat number between 100 and 200 times are mainly dementia; the clinical manifestations of patients with a repeat number above 200 times are mainly myasthenia; when the repeat number exceeds 300 times, the CpG island tends to be highly methylated and expression inhibited, thus producing asymptomatic carriers. In addition, there may be GGA insertions interrupting the GGC repeat sequence. Some scholars believe that this insertion is related to a weak dominant phenotype, and the clinical significance of single nucleotide site variations (SNVs) on the NOTCH2NLC gene remains to be explored.

[0005] Repeat primer PCR (RP-PCR) combined with capillary electrophoresis is a commonly used method for clinical detection of polynucleotide repeat mutations. It has the advantages of economy and speed, etc., but it has certain limitations in detecting the specific repeat number of the repeat sequence, the type of sequence insertion interruption and other mutation types.

[0006] Third-generation long-read sequencing (LRS) has unique advantages in analyzing complex gene structural variations, methylation research, tandem repeat regions, etc. due to its ultra-long read length. For tandem repeat regions, the targeted sequencing technology with CRISPR-Cas9 technology as the enrichment strategy requires high sample integrity and hundreds of times more input than this application; the enrichment cost is relatively high, and there is an off-target effect due to technical reasons, especially more serious off-target when targeting NOTCH2NLC with homologous sequences, that is, it is difficult to overcome the specificity problem, and additional data screening is required during analysis, ultimately resulting in increased waste of sequencing data and increased sequencing cost. Therefore, this method has limitations that are not conducive to clinical promotion in terms of sample DNA demand in micrograms, as well as cost and operation difficulty. Summary of the Invention

[0007] The present invention provides a primer combination, kit and system for detecting the NOTCH2NLC gene in order to solve the technical problems existing in the prior art. The present invention combines PCR-capillary electrophoresis (CE) with third-generation long-read sequencing (LRS) to provide more alternative ways for the detection of the NOTCH2NLC gene; compared with single RP-PCR, it can detect higher and more accurate GGC repeat numbers; lower proportion of chimeric mutations; it can detect more types of insertion and interruption types and other minor mutation types, which is beneficial to the exploration of its clinical significance; compared with whole-genome third-generation sequencing and CRISPR-Cas9 targeted enrichment third-generation sequencing, it has a DNA input amount far lower than their experimental requirements, a simpler and more economical operation process, and is more conducive to clinical application and promotion.

[0008] Among them, the PCR-capillary electrophoresis method includes two parts: long-range PCR (LR-PCR) and repeat primer PCR (RP-PCR), which can preliminarily screen positive samples with GGC expansion. According to the needs, the LR-PCR primer set can be used to further perform third-generation long-read sequencing (LRS) on the sample. The experiment only requires 20 ng of DNA and an average PCR targeted enrichment cost of less than 30 yuan per sample. Hundreds of samples can be detected simultaneously in one sequencing, greatly reducing the average sequencing cost. It can accurately detect GGC repeat numbers, insertions and interruptions such as GGA, and other minor mutations, and can also detect low-proportion chimeric mutations below the CE detection limit and higher GGC repeat numbers.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] The first aspect of the present invention provides a primer combination, which includes the following primer combinations:

[0011] The first set of long-range amplification primer combinations, the first set of long-range amplification primer combinations includes primers with nucleotide sequences shown in SEQ ID NO: 1 and 2;

[0012] The second set of long-range amplification primer combinations, wherein the second set of long-range amplification primer combinations includes primers with nucleotide sequences shown in SEQ ID NO: 6 and 7; and

[0013] The repeat amplification primer combination, wherein the repeat amplification primer combination includes primers with nucleotide sequences shown in SEQ ID NO: 3, 4, and 5.

[0014] It can be understood that when the amplification products of the above primer combinations need to be analyzed by capillary electrophoresis, according to the requirements of the detection platform, the 5' end of the forward primer (such as the primer with a nucleotide sequence shown in SEQ ID NO: 1, 6, or 3) needs to be modified with a fluorescent group, and the fluorescent group is a conventional option in the art, such as FAM, TET, VIC, HEX, etc.

[0015] In some embodiments of the present invention, the primer combination includes the first set of long-range amplification primer combinations.

[0016] In some embodiments of the present invention, the primer combination includes the second set of long-range amplification primer combinations.

[0017] In some embodiments of the present invention, the primer combination includes the repeat amplification primer combination.

[0018] In some embodiments of the present invention, the primer combination includes the first set of long-range amplification primer combinations and the repeat amplification primer combination.

[0019] In some embodiments of the present invention, the primer combination includes the second set of long-range amplification primer combinations and the repeat amplification primer combination.

[0020] The second aspect of the present invention provides a kit for detecting the NOTCH2NLC gene, and the kit includes the primer combination as described in the first aspect of the present invention.

[0021] In some embodiments of the present invention, in the first set of long-range amplification primer combinations, the working concentrations of the primers shown in SEQ ID NO: 1 and 2 are each independently 0.05 - 0.5 μM, for example, 0.35 μM; and / or, the working concentration ratio of the primers shown in SEQ ID NO: 1 and 2 is 0.5 - 1.5:1, for example, 1:1;

[0022] In the second set of long-range amplification primer combinations, the working concentrations of the primers shown in SEQ ID NO: 6 and 7 are each independently 0.05 - 0.5 μM, for example, 0.35 μM; and / or, the working concentration ratio of the primers shown in SEQ ID NO: 6 and 7 is 0.5 - 1.5:1, for example, 1:1;

[0023] In the primer combination for multiple amplification, the working concentrations of the primers shown in SEQ ID NO: 3, 4, and 5 are each independently 0.01 - 0.03 μM. For example, the working concentrations of the primers shown in SEQ ID NO: 3 and 4 are both 0.16 μM, and the working concentration of the primer shown in SEQ ID NO: 5 is 0.08 μM; and / or, the working concentration ratio of the primers shown in SEQ ID NO: 3, 4, and 5 is 1.5 - 2.5:1.5 - 2.5:1, for example, 2:2:1.

[0024] In some embodiments of the present invention, the kit further comprises one or more of the following amplification reagents:

[0025] (1) PCR polymerase; the PCR polymerase is preferably selected from the PCR polymerase with the manufacturer TOYOBO LIFE SCIENCE and the product number KFX - 201 and the PCR polymerase with the manufacturer Applied Biosystems and the product number 4398881;

[0026] (2) PCR enhancer; the PCR enhancer is preferably selected from one or more of betaine, DMSO, glycerol, 7 - deaza - dGTP, Mg 2+ , dNTP, and high - GC - content enhancer;

[0027] (3) PCR amplification buffer; and

[0028] (4) ddH 2 O.

[0029] In some embodiments of the present invention, the PCR polymerase is the PCR polymerase with the manufacturer TOYOBO LIFE SCIENCE and the product number KFX - 201, and the PCR enhancer is DMSO.

[0030] In some embodiments of the present invention, the PCR polymerase is the PCR polymerase with the manufacturer Applied Biosystems and the product number 4398881, and the PCR enhancer is betaine.

[0031] In some embodiments of the present invention, the kit comprises a PCR polymerase, a PCR enhancer, and the primer combination; when in use, the kit is preferably configured into the following reaction system: in a volume of 25 μL, the reaction system comprises 10 μL - 12.5 μL of the PCR polymerase with the manufacturer Applied Biosystems and the product number 4398881, 1 M - 2 M of betaine, 1.5 μL - 3.5 μL of the primer combination, and 1 μL - 2 μL of the nucleic acid sample to be tested. Among them, the primer combination is the first long-range amplification primer combination, the repeat amplification primer combination, or the second long-range amplification primer combination, the concentration of each primer is 0.05 - 0.5 μM, and the mass of DNA in the nucleic acid sample to be tested is 20 - 100 ng. An exemplary composition of the reaction system is as follows:

[0032]

[0033] In some embodiments of the present invention, the kit comprises a PCR amplification buffer, a PCR polymerase, a PCR enhancer, ddH 2 O, and the primer combination; when in use, the kit is preferably configured into the following reaction system: in a volume of 25 μL, the reaction system comprises 10 μL - 12.5 μL of the PCR amplification buffer, 3 μL - 5 μL of dNTP with a concentration of 2 mM, 0.5 μL - 1 μL of the PCR polymerase with the manufacturer TOYOBO LIFE SCIENCE and the product number KFX - 201, 2% - 12% (e.g., 10%) of DMSO, 1.5 μL - 2.5 μL of the primer combination, 1.5 μL - 3.5 μL of ddH 2 O, and 1 μL - 2 μL of the nucleic acid sample to be tested. Among them, the primer combination is the first long-range amplification primer combination, the repeat amplification primer combination, or the second long-range amplification primer combination, the concentration of each primer is 0.05 - 0.5 μM, and the mass of DNA in the nucleic acid sample to be tested is 20 - 100 ng. An exemplary composition of the reaction system is as follows:

[0034]

[0035]

[0036] In some embodiments of the present invention, the kit further comprises reagents for constructing a sequencing library.

[0037] In some embodiments of the present invention, the reagents include one or more of Barcode adapters, T4 DNA ligase buffer, ATP, T4 polynucleotide kinase, T4 DNA ligase, dNTP, and ddH 2 O.

[0038] In some embodiments of the present invention, the reagent is formulated into the following reaction system when constructing the library: in a volume of 10 μL, the reaction system for constructing the library includes 3-6 μL of PCR product, 1.5-3 μL of Barcode linker, 0.8-1.2 μL of 10×T4 DNA ligase buffer, 0.5-1.5 μL of ATP with a concentration of 10 mM, 0.1-0.5 μL of T4 polynucleotide kinase, 0.1-0.5 μL of T4 DNA ligase, and the rest is water. The composition of an exemplary reaction system for constructing the library is as follows:

[0039]

[0040] The reaction program corresponding to the above reaction system for constructing the library is: 37°C - 30 minutes; 20°C - 15 minutes; 65°C - 10 minutes.

[0041] In some embodiments of the present invention, the reaction system for constructing the library includes a blunt-end ligation library construction reaction system and a linker reaction system, wherein: in a volume of 10 μL, the blunt-end ligation library construction reaction system includes 4-7 μL of PCR product, 0.8-1.2 μL of 10×T4 DNA ligase buffer, 0.5-1.5 μL of ATP with a concentration of 10 mM, 0.1-0.5 μL of T4 polynucleotide kinase, 0.1-0.5 μL of T4 DNA ligase, 0.05-0.15 μL of dNTP with a concentration of 10 mM, and the rest is water; the product corresponding to the blunt-end ligation library construction reaction system is the first PCR product;

[0042] In a volume of 10 μL, the linker reaction system includes 4-7 μL of the first PCR product, 1.5-3 μL of Barcode linker, 0.8-1.2 μL of 10×T4 DNA ligase buffer, 0.1-0.5 μL of T4 polynucleotide kinase, 0.1-0.5 μL of T4 DNA ligase, and the rest is water.

[0043] The composition of an exemplary blunt-end ligation library construction reaction system is as follows in the table:

[0044]

[0045] The reaction program corresponding to the above blunt-end ligation library construction reaction system is: 37°C - 30 minutes; 65°C - 10 minutes.

[0046] The composition of an exemplary linker reaction system is as follows in the table:

[0047]

[0048] The reaction procedure corresponding to the above ligation reaction system is: 20°C - 15 minutes; store at 4°C.

[0049] The third aspect of the present invention provides the use of the primer combination as described in the first aspect of the present invention in the preparation of a kit for detecting the NOTCH2NLC gene.

[0050] In some embodiments of the present invention, the kit is the kit as described in the second aspect of the present invention.

[0051] The fourth aspect of the present invention provides a system for detecting the NOTCH2NLC gene, the system comprising:

[0052] A primary screening module, which receives the capillary electrophoresis results of the long-range amplification products and the capillary electrophoresis results of the repeat amplification products, calculates the number of GGC repeats based on the capillary electrophoresis results of the long-range amplification products, determines the positive and negative results based on the capillary electrophoresis results of the repeat amplification products, and determines the flow direction according to the following analysis results:

[0053] 1) When the capillary electrophoresis results of the long-range amplification products show three or more signal peaks with different GGC repeat numbers, it enters the sequencing module for third-generation long-read sequencing;

[0054] 2) When the number of GGC repeats is higher than the threshold, it enters the sequencing module for third-generation long-read sequencing;

[0055] 3) When the capillary electrophoresis results of the long-range amplification products only show one signal peak with the GGC repeat number within the normal range, but the capillary electrophoresis results of the repeat amplification products are positive, it enters the sequencing module for third-generation long-read sequencing;

[0056] 4) When the capillary electrophoresis results of the long-range amplification products show two or fewer signal peaks with different GGC repeat numbers, the capillary electrophoresis results of the repeat amplification products are negative, and the number of GGC repeats is less than the threshold, the result is directly output by the result output module;

[0057] A sequencing module, which performs third-generation long-read sequencing based on the DNA library constructed from the long-range amplification products; and

[0058] A result output module, which outputs the analysis results of the primary screening module or the sequencing results of the sequencing module;

[0059] Among them, the long-range amplification products include first long-range amplification products and / or second long-range amplification products. The first long-range amplification products are obtained based on the first set of long-range amplification primer combinations in the primer combination as described in the first aspect of the present invention; the repeated amplification products are obtained based on the repeated amplification primer combinations in the primer combination as described in the first aspect of the present invention; and the second long-range amplification products are obtained based on the second set of long-range amplification primer combinations in the primer combination as described in the first aspect of the present invention.

[0060] In the present invention, DNA libraries constructed based on the first long-range amplification products and DNA libraries constructed based on the second long-range amplification products can be used for sequencing simultaneously. The sequencing results of different DNA libraries are analyzed respectively, and data with better quality control indicators are selected as the basis for obtaining the results. It can be understood that the above quality control indicators are determined in accordance with the basic requirements of bioinformatics analysis in the art, comprehensively considering sequencing depth, sequencing coverage, base quality (Q30 ratio), and adapter sequence contamination.

[0061] In the present invention, when the result output module outputs the results of third-generation long-read sequencing, the output results include, but are not limited to, the measured sequences, GGC repeat numbers, SNVs, and GGA insertions; when the result output module outputs the capillary electrophoresis results, the output results include GGC repeat numbers and positive / negative.

[0062] In some embodiments of the present invention, when the number of third-generation long-read sequencing reads is greater than 1000, the results are directly output according to the sequencing result sequences; when the number of third-generation long-read sequencing reads is less than 1000, the experimental steps need to be reviewed and re-detected.

[0063] In some embodiments of the present invention, the first long-range amplification products, the second long-range amplification products, the repeated amplification products, and / or the DNA libraries are obtained by using the kit as described in the second aspect of the present invention.

[0064] In some embodiments of the present invention, the first long-range amplification products and / or the second long-range amplification products are obtained through the following amplification program: pre-denaturation at 98°C for 10 minutes; denaturation at 98°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 4 minutes, for 32 cycles; final extension at 72°C for 10 minutes.

[0065] In some embodiments of the present invention, the repeated amplification products are obtained through the following amplification program: pre-denaturation at 98°C for 10 minutes; denaturation at 98°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 68°C for 4 minutes, for 35 cycles; final extension at 68°C for 10 minutes.

[0066] In some embodiments of the present invention, the number of GGC repeats is calculated by the following formula: (fragment length - 188bp) / 3X, where "X" is the capillary instrument coefficient. Since the number of nucleotides in the repeat sequence GGC is 3, generally X is 1; however, there are certain deviations for different capillary instruments. For example, for the instrument used in the examples of the present invention, 3X is 2.6828, that is, the number of GGC repeats n = (fragment length - 188bp) / 2.6828.

[0067] The "threshold" as described in the present invention refers to the value for judging the full mutation of the NOTCH2NLC gene in the test sample based on the number of GGC repeats in the medical field, which can be adjusted according to the actual diagnostic criteria. In some embodiments of the present invention, the threshold is 65.

[0068] The "normal range" as described in the present invention refers to the data range for judging the normality of the test sample based on the number of GGC repeats in the medical field, which can be adjusted according to the actual diagnostic criteria. In some embodiments of the present invention, the normal range of the number of GGC repeats is ≤38.

[0069] In some embodiments of the present invention, when the number of GGC repeats is less than 39, it is judged as normal; when the number of GGC repeats is 39 - 65, it is judged as a medium mutation; when the number of GGC repeats is greater than 65, it is judged as a full mutation.

[0070] In some embodiments of the present invention, the positive or negative is determined based on the capillary electrophoresis result of the repeat amplification product. When there are continuous ladder-like signals and the signal peak extends beyond the abscissa 280, or when the number of signal peaks exceeds 38, it is judged as a positive result; otherwise, it is judged as a negative result.

[0071] The fifth aspect of the present invention provides a method for detecting the NOTCH2NLC gene, the method comprising detecting a test sample using the kit as described in the second aspect of the present invention or the system as described in the fourth aspect of the present invention and obtaining a detection result.

[0072] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0073] The reagents and raw materials used in the present invention are all commercially available.

[0074] The positive and progressive effects of the present invention are as follows:

[0075] The primer combination provided by the present invention is obtained through specific screening. The kit and system based on this primer combination can detect a wider range of mutation types, can detect SNV, insertions and disruptions such as GGA, as well as lower proportions of chimeras and higher numbers of GGC repeats, and have simple operations and low detection costs. Description of the Drawings

[0076] Figure 1 shows the capillary electrophoresis results of the PCR amplification products using

[0076] and Figure 1 as LR-PCR amplification primers and GXL DNA Polymerase as the PCR polymerase. shows the capillary electrophoresis results of the PCR amplification products using

[0076] and Figure 1 as LR-PCR amplification primers and Advantage GC 2 PCR Kit as the PCR polymerase.

[0077] Figure 2 shows the capillary electrophoresis results of the PCR amplification products using

[0076] and Figure 1 as LR-PCR amplification primers and Expand Long Template PCR Syst as the PCR polymerase.

[0078] Figure 3 shows the capillary electrophoresis results of the PCR amplification products using and as LR-PCR amplification primers and Max Master Mix as the PCR polymerase.

[0079] Figure 4 shows the capillary electrophoresis results of the PCR amplification products using and as LR-PCR amplification primers and AmpliTaq Gold 360 MasterMix as the PCR polymerase. shows the capillary electrophoresis results of the PCR amplification products using and as LR-PCR amplification primers and AmpliTaq Gold 360 MasterMix as the PCR polymerase.

[0080] Figure 5 shows the capillary electrophoresis results of the PCR amplification products using and as LR-PCR amplification primers and AmpliTaq Gold 360 MasterMix as the PCR polymerase.

[0081] Figure 6 shows the capillary electrophoresis results of the PCR amplification products using and

[0082] as LR-PCR amplification primers and AmpliTaq Gold 360 MasterMix as the polymerase.

[0082] Figure 7 shows the gel electrophoresis results of the PCR amplification products using and

[0083] as LR-PCR amplification primers and AmpliTaq Gold 360 MasterMix as the polymerase.

[0083] Figures 8 - 12 Comparison shows the capillary electrophoresis results of the PCR amplification products using and

[0082] as LR-PCR amplification primers and AmpliTaq Gold 360 Master Mix as the polymerase under different enhancers and amplification conditions.

[0084] Figure 13 and 14The comparison shows the capillary electrophoresis results of PCR amplification products with the 1st and 2nd as LR-PCR amplification primers, using KOD FX Neopolymerase as the polymerase under different enhancers and amplification conditions.

[0085] Figure 15 It shows the capillary electrophoresis results of PCR amplification products with the 1st and 2nd as LR-PCR amplification primers, using Q5 Hot start high-Fidelity 2X Master as the polymerase.

[0086] Figure 16 It shows the capillary electrophoresis results of PCR amplification products with the 1st and 2nd as LR-PCR amplification primers, using LongAmp Taq 2X MasterMix as the polymerase.

[0087] Figure 17 It shows the capillary electrophoresis results of PCR amplification products with the 1st and 2nd as LR-PCR amplification primers, using Max Master Mix as the polymerase.

[0088] Figure 18 It shows the capillary electrophoresis results of PCR amplification products with the 8th, 20th and 25th as RP-PCR amplification primers.

[0089] Figure 19 It shows the capillary electrophoresis results of PCR amplification products with the 3rd, 4th and 5th as RP-PCR amplification primers.

[0090] Figure 20 It shows the positive result of the LR-PCR detection of the S2 sample.

[0091] Figure 21 It shows the negative result of the LR-PCR detection of the S11 sample.

[0092] Figure 22 It shows the positive result of the RP-PCR detection of the S2 sample.

[0093] Figure 23 It shows the negative result of the RP-PCR detection of the S11 sample.

[0094] Figure 24 It shows the change graph of the number of reads corresponding to the GGC repeat number of the S6 sample detected by the detection method corresponding to the TA library construction reaction system.

[0095] Figure 25Shows the cumulative distribution change of the data corresponding to the GGC repeat number of sample S6 detected by the detection method using the TA library construction reaction system.

[0096] Figure 26 Shows the change graph of the number of reads corresponding to the GGC repeat number of sample S1 detected by the detection method using the blunt-end ligation library construction reaction system.

[0097] Figure 27 Shows the cumulative distribution change of the data corresponding to the GGC repeat number of sample S1 detected by the detection method using the blunt-end ligation library construction reaction system.

[0098] Figure 28 A comparison shows the SNVs detected by the third-generation sequencing detection method for some samples S1, S2, and S3.

[0099] Figure 29 A comparison shows the SNVs detected by the third-generation sequencing detection method for some samples S7 and S8.

[0100] Figure 30 Shows the relationship graph between the sequencing length and the base data volume under the ONT platform. Specific implementation manners

[0101] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0102] 1. Some primer sequences used in the examples (as shown in Table 1)

[0103] Table 1 Sequence and final concentration range of primers

[0104]

[0105] 2. The PCR polymerases used in the following examples (as shown in Table 2)

[0106] Table 2 PCR polymerases

[0107]

[0108] 3. The PCR enhancers used in the following examples (as shown in Table 3)

[0109] Table 3 PCR enhancers

[0110] Material Name Manufacturer Final Concentration Range B0300 - 5VL Betaine Sigma - Aldrich 1M - 2M D2650 DMSO Solution (≥99.7%) Sigma - Aldrich 2％-12％ G5516 - 100ML Glycerol Sigma - Aldrich 2％-6％ 7 - deaza - dGTP NEW ENGLAND BIOLABS (NEB) 200μM - 600μM <![CDATA[MgCl 2 25mM]]> Sangon Biotech (Shanghai) Co., Ltd. 1.5 - 3mM dNTP (4 - tube set, 100mM) Shanghai Beyotime Biotechnology Co., Ltd. 200μM - 600μM GC enhancer ABI (Applied Biosystems) 5％-20％

[0111] Example 1: LR-PCR primer screening and amplification condition screening

[0112] I. Primer Screening (Tested by Capillary Electrophoresis and Gel Electrophoresis)

[0113] 1. Samples

[0114] Select clinically diagnosed samples that have undergone skin or lip gland pathological examinations to confirm the presence of intranuclear inclusions and have completed cranial MRI examinations showing characteristic imaging changes; normal samples are used as negative cases. Extract sample nucleic acids by the magnetic bead method, and measure that OD260 / OD280 is between 1.8 and 2.0, and the DNA concentration is greater than 20 ng / μL.

[0115] 2. Primer Design

[0116] First, select the 12th and 13th in Table 1 above as LR-PCR amplification primers, and use GXL DNA Polymerase, Advantage GC 2 PCR Kit, and Expand Long Templ PCR Syst as PCR polymerases, and add DMSO solution and MgCl 2 as PCR enhancers, set the annealing temperature to 64°C, and select the optimal denaturation and extension temperatures of the polymerase according to the reagent instructions.

[0117] The specific reaction system is as follows:

[0118]

[0119]

[0120] The specific amplification program is as follows:

[0121] Pre-denature at 95°C for 3 minutes; 95°C - 30 seconds, 64°C - 30 seconds, 68°C - 1 minute, 35 cycles; extend at 68°C for 10 minutes. Non-specific multiple band amplifications appear in the results of all three reagent schemes (such as Figures 1 - 3 ), indicating insufficient primer specificity.

[0122] Further comparative analysis based on the UCSC hg38 database found that there are only individual base differences between NOTCH2NLC, NOTCH2NLA, and NOTCH2NLB near the GGC repeat amplification region. Therefore, special attention should be paid to specificity in the primer selection position to distinguish from NOTCH2NLA and NOTCH2NLB, and the primers should be located at specific sites as much as possible, and primers with ARMS-PCR properties are designed.

[0123] Based on the above analysis, multiple primers were redesigned for experimental attempts. For example, the 3' end of the primer was adjusted to be located at a specific site of the NOTCH2NLC gene sequence, and primer F (forward primer) was designed. Typical primers F are primers 1, 7, 8, 10 and 16 in Primer Sequence Table 1; primer R (reverse primer) was designed. Typical primers R are primers 2, 6, 9, 11, 17 and 22 in Primer Sequence Table 1, among which primers 1, 6, 8, 9 and 11 modified the third base at the 3' end of the sequence to form mismatch primers of ARMS-PCR nature to reduce the generation of non-specific products. Primer F and primer R were combined in sequence for testing.

[0124] Moreover, considering the specificity, the adjustment of primers is subject to certain restrictions. Improving the specificity will inevitably reduce the amplification efficiency, which makes the highly repetitive GC-rich regions that are already difficult to amplify even more difficult to amplify. For example, if the 8th and 9th items in Table 1 are LR-PCR amplification primers, The enzyme mixture of Max Master Mix and AmpliTaq Gold360Master Mix is ​​a polymerase, and betaine, DMSO solution and 7-deaza-dGTP enhancer are added to further improve specificity and amplification efficiency. The annealing temperature is 64°C, and the optimal denaturation and extension temperature of the polymerase is selected according to the reagent instructions.

[0125] The specific reaction system is as follows:

[0126] Reagent Volume 2×PCR Enzyme Mix 12.5μL 5M Betaine 5μL DMSO 1.25μL 5mM 7 - deaza - dGTP 1μL 5μM Primer Mix 2μL DNA (20 - 100ng) 1μL

[0127] The specific reaction procedure is as follows:

[0128] Pre-denaturation at 95°C for 10 minutes; 35 cycles of 95°C for 30 seconds, 64°C for 30 seconds, and 72°C for 1 minute; extension at 72°C for 10 minutes.

[0129] The results showed low amplification efficiency, such as low signal and obvious signal decrease of alleles with larger GGC repeat numbers (results such as Figure 4 and 5 ). The remaining combinations all had no amplification signals and mixed peaks.

[0130] According to the above method, the test was carried out respectively, and according to the test results, the combination of the first and second primer sequences in the primer sequence table 1 was finally determined ( Figure 6 ), the combination of Article 6 and Article 7 is optimal ( Figure 7) Among them, the product amplified by the combination of the 1st and 2nd primers is suitable for capillary electrophoresis and third-generation sequencing platform detection; the product amplified by the combination of the 6th and 7th primers has a length exceeding 1 Kb. In terms of detecting SNV, the combination of the 6th and 7th primers is more suitable for third-generation sequencing platform detection than the combination of the 1st and 2nd primers.

[0131] II. Reagent combination and amplification condition screening:

[0132] Using the combination of the 1st and 2nd in Primer Sequence Table 1, test the reagent combination and PCR amplification conditions. The polymerases selected are Q5 Hot start high-Fidelity 2X Master, LongAmp Taq 2X Master Mix, MaxMaster Mix, KOD FX Neo polymerase, and AmpliTaq Gold 360Master Mix, combined with enhancers such as betaine, DMSO solution, 7-deaza-dGTP, and GC enhancer. Set the annealing temperatures to 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, and the denaturation temperatures to 94°C, 95°C, 96°C, 97°C, 98°C. Select the optimal extension temperature of the polymerase according to the reagent instructions.

[0133] According to the following amplification system and different polymerases and enhancers in Table 4: 1 μL of sample DNA (20 - 100 ng), 2 μL of 5 μM primer mixture, polymerase, enhancer, dNTPs, Buffer, and ddH 2 O, and the total volume of the system is 25 μL.

[0134] According to the following amplification program and different temperature conditions in Table 4 for condition screening: pre-denaturation for 10 minutes; denaturation for 30 seconds, annealing for 30 seconds, extension for 4 minutes, 32 cycles; final extension for 10 minutes.

[0135] The specific test conditions for each group are shown in Table 4 below:

[0136] Table 4 Test conditions and results for each group

[0137]

[0138] In addition to the screening conditions listed above, the inventor also tried experimental explorations in combination with the above reaction system and other amplification programs in the early stage. Typical results are as Figures 15 - 17 shown, among which, Figure 15 is the result of the combination of Q5 Hot start high-Fidelity 2XMaster polymerase and betaine, denaturing at 98°C, annealing at 65°C, and extending at 72°C.Figure 16 For The result of the combination of Max Master Mix polymerase and betaine, denatured at 98 °C, annealed at 65 °C, and extended at 72 °C, Figure 17 is the result of the combination of LongAmp Taq 2X Master Mix polymerase and betaine, denatured at 98 °C, annealed at 65 °C, and extended at 72 °C. A certain degree of messy signals are generated in all the above three combination methods.

[0139] The experimental results show that under the conditions of using AmpliTaq Gold 360 Master Mix as the polymerase and betaine as the enhancer, with the denaturation and extension conditions unchanged, reducing the annealing temperature can effectively improve the amplification efficiency. However, reducing the annealing temperature will also lead to messy signals and interfere with the generation of signals (such as Figures 9 - 11 ).

[0140] The addition of the enhancer will cause changes in the annealing temperature conditions. Different enhancers have different effects on the annealing temperature. It can be seen from the above results that the combination of multiple enhancers makes the amplification conditions complex, easily leading to reaction failure and no signal generation. Compared with the combination of multiple enhancers, a single enhancer with a higher concentration is more conducive to the stability of the system, and the most suitable enhancer for different polymerases also has differences.

[0141] Based on the comprehensive results of all screening tests, it is finally determined that an amplification system with KOD FX Neo polymerase as the polymerase and 10% DMSO solution as the enhancer, or with AmpliTaq Gold 360 Master Mix as the polymerase and 1.6 M betaine as the enhancer has better effects when denatured at 98 °C for 10 minutes and annealed at 65 °C for 30 seconds (i.e., groups 7 and 9 in Table 4 above).

[0142] Example 2: Capillary electrophoresis detection of LR-PCR amplification products

[0143] 1. Prepare the primer mixture: Mix SEQ ID NO:1 and SEQ ID NO:2 in equal volume at a primer concentration of 5 μM to form an LR-PCR primer mixture, where the 5' end of the SEQ ID NO:1 primer is fluorescently modified with FAM;

[0144] 2. Configure the PCR reaction system according to the following table:

[0145] Reagent Volume AmpliTaq Gold 360Master Mix (ABI, 4398881) 12.5μL Betaine (Sigma - Aldrich, B0300 - 5VL) 8μL 5μM Primer Mix 3.5μL DNA (20 - 100ng) 1μL

[0146] 3. Perform PCR amplification with pre-denaturation at 98 °C for 10 minutes; denaturation at 98 °C for 30 seconds, annealing at 65 °C for 30 seconds, extension at 72 °C for 4 minutes, for 32 cycles; and final extension at 72 °C for 10 minutes.

[0147] 4. After the reaction is completed, take 1 μL of the above amplification product respectively, mix it with 9.8 μL of deionized formamide and 0.2 μL of the molecular weight internal standard Liz500, denature at 95 °C for 5 minutes, and then cool on ice for 3 minutes. Perform capillary electrophoresis on ABI 3500, with an injection voltage of 1.6 kV for 15 seconds; a running voltage of 19.5 kV for 1330 seconds. Analyze the electrophoresis results using Genemapper 5.

[0148] 5. Result analysis: Count the fragment length, and calculate the number of GGC repeats n according to the following formula:

[0149] n = (fragment length - 188 bp) / 2.6828.

[0150] Example 3: RP-PCR primer screening

[0151] The samples in this example are the same as those in Example 1.

[0152] On the basis of the amplification conditions screened in Example 2, through preliminary experiments, further select the 3rd to 5th, 8th, 20th, and 25th primers in Primer Sequence Table 1 as the primers for RP-PCR, and adopt the following reaction program: denature at 98 °C for 30 seconds, anneal at 65 °C for 30 seconds, extend at 68 °C for 4 minutes, for 35 cycles; finally extend at 68 °C for 10 minutes to perform PCR amplification; keep other conditions unchanged for RP-PCR primer screening, and the 5' ends of the 3rd and 8th primers are modified with FAM fluorescence.

[0153] The results are as Figures 18 - 19 shown. It can be seen from the results that when the 3rd, 4th, and 5th primers in Primer Sequence Table 1 are used as the primers for RP-PCR, the amplification products have no obvious miscellaneous peaks and have clear main peaks (signal peaks with the maximum number of repeats), which is more conducive to the interpretation of the results.

[0154] Example 4: Capillary electrophoresis detection of RP-PCR amplification products

[0155] 1. Prepare the primer mixture: SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 are used as the primers for RP-PCR, with a primer concentration of 5 μM, and make a primer mixture according to a volume ratio of 2:2:1. The 5' end of the SEQ ID NO:3 primer is modified with FAM fluorescence;

[0156] 2. According to the different primer mixtures, configure the PCR reaction system as follows:

[0157] Reagent Volume 2×PCR Buffer (Toyobo, KFX - 201) 12.5μL 2mM dNTPs (Toyobo, KFX - 201) 5μL 5μM Primer Mix 2μL KOD FX Neo (Toyobo, KFX - 201) 0.5μL DMSO (Sigma - Aldrich, D2650) 2.5μL <![CDATA[ddH 2 O]]> 1.5μL DNA (20 - 100ng) 1μL

[0158] 3. Pre-denature at 98°C for 10 minutes; denature at 98°C for 30 seconds, anneal at 65°C for 30 seconds, extend at 68°C for 4 minutes, for 35 cycles; finally extend at 68°C for 10 minutes for PCR amplification.

[0159] 4. After the reaction, take 1 μL of the above amplification product respectively, mix it with 9.8 μL of deionized formamide and 0.2 μL of molecular weight internal standard Liz500, denature at 95°C for 5 minutes and then cool on ice for 3 minutes. Perform capillary electrophoresis on ABI 3500, injection voltage 1.6 kV, for 15 seconds; running voltage 19.5 kV, for 1330 seconds. Analyze the electrophoresis results using Genemapper 5.

[0160] 5. Result analysis:

[0161] Judge positive and negative according to the capillary electrophoresis results. When continuous ladder-like signals appear and the signal peak extends beyond the abscissa 280, or when the number of signal peaks exceeds 38, it is judged as a positive result; otherwise, it is judged as a negative result.

[0162] Example 5: PCR Targeted Amplification for Pacbio Third-generation Sequencing Detection

[0163] 1. Prepare primer mixtures: Mix SEQ ID NO:1 and SEQ ID NO:2 in equal volumes to form a PCR primer mixture, and mix SEQ ID NO:6 and SEQ ID NO:7 in equal volumes to form a PCR primer mixture. The primer concentrations before mixing are both 5 μM, and place them in different PCR tubes to form the first amplification system and the second amplification system respectively;

[0164] 2. Configure the PCR reaction system according to the following table:

[0165] Reagent Volume AmpliTaq Gold 360Master Mix (ABI, 4398881) 12.5μL Betaine (Sigma - Aldrich, B0300 - 5VL) 8μL 5μM Primer Mix 3.5μL DNA (20 - 100ng) 1μL

[0166] 3. Pre-denature at 98°C for 10 minutes; denature at 98°C for 30 seconds, anneal at 65°C for 30 seconds, extend at 72°C for 4 minutes, for 32 cycles; finally extend at 72°C for 10 minutes for the amplification program for PCR amplification.

[0167] 4. Purify the PCR amplification product twice with 1×Ampure PB magnetic beads and 80% ethanol according to the instructions, elute the DNA with 10 μL of elution buffer, and the eluted product is used for the TA library construction reaction system (the specific composition is as follows in the table):

[0168] Reagent Volume Eluted Product 4μL Barcode Adapter 2.5μL 10×T4 DNA Ligase Buffer (Enzymatics, B6030) 1μL ATP (10mM) (Enzymatics, B6030) 1μL T4 Polynucleotide Kinase (Enzymatics, Y9040L) 0.3μL T4 DNA Ligase (Enzymatics, L6030 - HC - L) 0.3μL <![CDATA[ddH 2 O]]> 0.9μL

[0169] 5. Run the PCR program at 37°C for 30 minutes; 20°C for 15 minutes; 65°C for 10 minutes.

[0170] 6. After the reaction, add 0.5 μL Exonuclease III (NEB, M0206L) and 0.5 μL Exonuclease VII (NEB, M0379L) to the product and react at 37°C for one hour. Purify twice with 1×Ampure PB magnetic beads and 80% ethanol according to the instructions, and finally elute the DNA with 15 μL elution buffer. The eluted product is the target DNA sequencing library.

[0171] 7. The mixed library was prepared according to the instructions of Sequel Sequencing Kit (Pacific Biosciences, 101-310-500) and sequenced on Sequel II e (Pacific Biosciences).

[0172] 8. Analyze the offline data and output the sequencing results.

[0173] Example 6: PCR targeted amplification Pacbio third-generation sequencing detection

[0174] 1. Prepare primer mixture: same as Example 5.

[0175] 2. Configure the PCR reaction system: same as in Example 5.

[0176] 3. PCR amplification: same as Example 5.

[0177] 4. Library construction: The PCR amplification product was purified twice with 1×Ampure PB magnetic beads and 80% ethanol according to the instructions, and the DNA was eluted with 10 μl elution buffer. The eluted product was used for blunt-end ligation library construction reaction system (its composition is shown in the following table):

[0178] Reagent Volume Eluted Product 5μL 10×T4 Polynucleotide Kinase Buffer (Enzymatics, B9040) 1 μL ATP (10 mM) (Enzymatics, B6030) 1 μL T4 Polynucleotide Kinase (Enzymatics, Y9040L) 0.3 μL T4 DNA Polymerase (Enzymatics, P7080L) 0.3 μL dNTP (10 mM) 0.1 μL <![CDATA[ddH 2 O]]> 2.3 μL

[0179] 5. Run the PCR program at 37°C for 30 minutes and 65°C for 10 minutes.

[0180] 6. The reaction product was purified twice with 1×Ampure PB magnetic beads and 80% ethanol according to the instructions, and the DNA was eluted with 10 μL elution buffer. The system was configured as shown in the following table:

[0181] Reagent Volume Purified Product 5 μL Barcode Adapter 2.5 μL 10×T4 DNA Ligase Buffer (Enzymatics, B6030) 1 μL T4 DNA Ligase (Enzymatics, L6030-HC-L) 0.3 μL <![CDATA[ddH 2 O]]> 1.2 μL

[0182] 7. Incubate at 20℃ for 15 minutes and store at 4℃. Run the PCR program.

[0183] 8. After the reaction is completed, add 0.5 μL of Exonuclease III (NEB, M0206L) and 0.5 μL of Exonuclease VII (NEB, M0379L) to the product and react at 37 °C for one hour. Purify twice according to the instructions using 1× Ampure PB magnetic beads and 80% ethanol. Finally, elute the DNA with 15 μL of elution buffer, and aspirate the eluted product, which is the target DNA sequencing library.

[0184] 9. Prepare the library for sequencing on the machine according to the reagent instructions of the Sequel Sequencing Kit (Pacific Biosciences, 101-310-500), and sequence it on the Sequel Ⅱe (Pacific Biosciences).

[0185] 10. Analyze the data downloaded from the machine and output the sequencing results.

[0186] Example 7: PCR Targeted Amplification and ONT Third-Generation Sequencing Detection

[0187] 1. Prepare the primer mixture: same as Example 5;

[0188] 2. Configure the PCR reaction system: same as Example 5.

[0189] 3. PCR amplification: same as Example 5.

[0190] 4. TA library construction: same as Example 5.

[0191] 5. PCR program: same as Example 5.

[0192] 6. Library purification: same as Example 5.

[0193] 7. Prepare the library for sequencing on the machine according to the reagent instructions of the Ligation sequencing kit (ONT, SQK-LSK110), and sequence it on the MinION Mk1B (ONT).

[0194] 8. Analyze the data downloaded from the machine and output the sequencing results.

[0195] Verification Example

[0196] Thirty samples were selected (among them: 10 clinically diagnosed samples, which had undergone skin or lip gland pathological examinations, and nuclear inclusions were confirmed to exist, and cranial MRI examinations were completed, showing characteristic imaging changes; 10 suspected NIID patients with symptoms such as cognitive impairment, but without pathological diagnosis; 10 control samples of non-NIID patients taken during the same period). The detection methods of Examples 2 and 4 were used for primary screening. The results of 10 samples in the confirmed group and 4 samples in the suspected group were positive (capillary electrophoresis detection of LR-PCR amplification products showed that the number of GGC repeats was greater than 39, and RP-PCR amplification product capillary electrophoresis detection signals showed continuous stepped signals and the signal peaks extended beyond the abscissa of 280, or the number of signal peaks exceeded 38).

[0197] The above 14 samples with positive primary screening results and 1 randomly selected normal group sample were detected using the detection method of Example 5. Comparing the part of the GGC repeat number, the results of capillary electrophoresis and third-generation sequencing were consistent. Since the third-generation sequencing results included GGA insertions, while capillary electrophoresis only counted continuous GGC repeats, the third-generation sequencing results were about 4 repeats more than the capillary electrophoresis results in terms of the repeat number. When the number of GGC repeats was high, choosing different main signal peaks for data processing would result in a difference of 2 - 9 repeats.

[0198] For samples S7 and S8, due to the excessively high number of GGC repeats, exceeding 110, capillary electrophoresis could only perform qualitative analysis on them, while third-generation sequencing was more accurate in detecting the repeat number and could accurately detect the repeat number, which was beneficial for accurate clinical judgment. Sample S9 was not detected to have chimerism in LR-PCR, but low-proportion chimerism with more than 200 repeats was detected in the third-generation sequencing results.

[0199] In summary, in terms of the number of GGC repeats, capillary electrophoresis is suitable for the detection and qualitative detection of low-repeat samples, while third-generation sequencing can detect GGC repeats exceeding 200 times and lower-proportion chimerism, which is beneficial for improving the detection accuracy and contributing to clinical diagnosis.

[0200] The detection results of the above 14 samples with positive primary screening results and 1 randomly selected normal group sample are shown in Table 5 below and Figure 20 - Figure 30 . Among them:

[0201] Figure 20 and Figure 21 respectively show the positive and negative results of the LR-PCR detection of sample S2 and sample S11. The difference between the positive and negative sites is reflected in the abscissa fragment length.

[0202] Figure 22 and Figure 23The positive and negative results of RP-PCR detection of S2 sample and S11 sample are shown respectively. The differences between the positive sites and the negative sites are reflected in the fragment length of the abscissa and the number of signal peaks.

[0203] Figure 24 The change graph of the number of reads corresponding to the GGC repeat number of S6 sample detected by the detection method corresponding to the TA library construction reaction system (Example 5) is shown. Figure 26 The change graph of the number of reads corresponding to the GGC repeat number of S1 sample detected by the detection method corresponding to the blunt-end ligation library construction reaction system (Example 6) is shown, which also includes GGA insertion. The specific insertion position can be directly obtained from the sequencing sequence.

[0204] Figure 25 The cumulative distribution change of the data of S6 sample corresponding to the GGC repeat number detected by the detection method corresponding to the TA library construction reaction system (Example 5) is shown. Figure 27 The cumulative distribution change of the data of S1 sample corresponding to the GGC repeat number detected by the detection method corresponding to the blunt-end ligation library construction reaction system (Example 6) is shown.

[0205] Figure 28 and Figure 29 The SNVs detected by the third-generation sequencing detection method for some samples are shown for comparison Figure 28 (S1, S2, S3 samples): IGV result maps of NC_000001.11:g.149391335C>A and NC_000001.11:g.149391336G>A, Figure 29 (S7, S8 samples): IGV result map of NC_000001.11:g.149390838C>A).

[0206] Figure 30 The relationship graph between the sequencing length and the base data volume of S7, S8, S9, S10, and S11 samples sequenced on the ONT platform (corresponding to the detection method of Example 7) is shown.

[0207] Table 5 Results of each detection method

[0208]

[0209] Note: In the "LR-PCR result" and "third-generation sequencing result", the number before the semicolon is the GGC repeat number of the first allele, and the number after the semicolon is the GGC repeat number of the second allele. For example, "19;110" means that the GGC repeat number of the first allele is 19, and the GGC repeat number of the second allele is 110 times. If there is a chimera, different GGC repeat numbers are listed after the semicolon with a comma.

[0210] The above embodiments are only descriptions of the preferred embodiments of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, various modifications and improvements made by those skilled in the art to the technical solutions of the present invention shall still be covered by the claims of the present invention.

Claims

1. A primer combination, characterized in that: Includes the following primer combinations: A first set of long-range amplification primer combinations, wherein the first set of long-range amplification primer combinations comprises primers having nucleotide sequences as shown in SEQ ID NOs: 1 and 2; A second set of long-range amplification primer combinations, wherein the second set of long-range amplification primer combinations comprises primers having nucleotide sequences as shown in SEQ ID NOs: 6 and 7; and A repeat amplification primer combination, wherein the repeat amplification primer combination comprises primers whose nucleotide sequences are shown in SEQ ID NO: 3, 4 and 5.

2. The primer combination according to claim 1, characterized in that The primer combination includes the first set of long-range amplification primer combination; or The primer combination includes the second set of long-range amplification primer combination; or The primer combination includes the repeat amplification primer combination.

3. A kit for detecting NOTCH2NLC gene, characterized in that: The kit comprises the primer combination as claimed in claim 1 or 2; Preferably, In the first set of long-range amplification primer combinations, the working concentrations of the primers shown in SEQ ID NOs: 1 and 2 are independently 0.05-0.5 μM, for example, 0.35 μM; and / or, the working concentration ratio of the primers shown in SEQ ID NOs: 1 and 2 is 0.5-1.5:1, for example, 1:1; In the second set of long-range amplification primer combinations, the working concentrations of the primers shown in SEQ ID NOs: 6 and 7 are independently 0.05-0.5 μM, for example, 0.35 μM; and / or, the working concentration ratio of the primers shown in SEQ ID NOs: 6 and 7 is 0.5-1.5:1, for example, 1:1; In the repeated amplification primer combination, the working concentrations of the primers shown in SEQ ID NOs: 3, 4 and 5 are each independently 0.01-0.03 μM, for example, the working concentrations of the primers shown in SEQ ID NOs: 3 and 4 are both 0.16 μM, and the working concentration of the primer shown in SEQ ID NO: 5 is 0.08 μM; and / or, the working concentration ratio of the primers shown in SEQ ID NOs: 3, 4 and 5 is 1.5-2.5:1.5-2.5:1, for example, 2:2:

1.

4. The kit according to claim 3, characterized in that The kit may further comprise one or more of the following amplification reagents: (1) PCR polymerase; the PCR polymerase is preferably selected from the PCR polymerase produced by TOYOBO LIFE SCIENCE with a product number of KFX-201 and the PCR polymerase produced by Applied Biosystems with a product number of 4398881; (2) PCR enhancer; the PCR enhancer is preferably selected from betaine, DMSO, glycerol, 7-deaza-dGTP, Mg 2+ , one or more of dNTPs and high GC content enhancers; (3) PCR amplification buffer; and (4) ddH2O; Preferably, the PCR polymerase is a PCR polymerase produced by TOYOBO LIFE SCIENCE with a product number of KFX-201, and the PCR enhancer is DMSO; or The PCR polymerase is produced by Applied Biosystems with a product number of 4398881, and the PCR enhancer is betaine.

5. The kit according to claim 4, characterized in that The kit includes a PCR polymerase, a PCR enhancer and the primer combination; the kit is preferably configured into the following reaction system when used: based on a volume of 25 μL, the reaction system includes 10 μL-12.5 μL of a PCR polymerase produced by Applied Biosystems with a product number of 4398881, 1 M-2 M betaine, 1.5 μL-3.5 μL of the primer combination, and 1 μL-2 μL of a nucleic acid sample to be tested; Or, the kit includes PCR amplification buffer, PCR polymerase, PCR enhancer, ddH2O and the primer combination; the kit is preferably configured into the following reaction system when used: based on a volume of 25 μL, the reaction system includes 10 μL-12.5 μL of PCR amplification buffer, 3 μL-5 μL of dNTP with a concentration of 2 mM, 0.5 μL-1 μL of PCR polymerase with a product number of KFX-201 manufactured by TOYOBO LIFE SCIENCE, 2%-12% of DMSO, 1.5 μL-2.5 μL of the primer combination, 1.5 μL-3.5 μL of ddH2O and 1 μL-2 μL of the nucleic acid sample to be tested; Wherein, the primer combination is the first group of long-range amplification primer combination, the repeated amplification primer combination or the second group of long-range amplification primer combination, the concentration of each primer is 0.05-0.5 μM, and the mass of DNA in the nucleic acid sample to be tested is 20-100 ng.

6. The kit according to any one of claims 3 to 5, characterized in that The kit also includes reagents for constructing a library for sequencing; Preferably, the reagents include one or more of a Barcode adapter, a T4 DNA ligase buffer, ATP, a T4 polynucleotide kinase, a T4 DNA ligase, dNTP and ddH2O.

7. The kit according to claim 6, characterized in that The reagents are prepared into the following reaction system when constructing the library: Based on a volume of 10 μL, the reaction system for constructing the library includes 3-6 μL of PCR product, 1.5-3 μL of Barcode adapter, 0.8-1.2 μL of 10×T4 DNA ligase buffer, 0.5-1.5 μL of 10 mM ATP, 0.1-0.5 μL of T4 polynucleotide kinase, 0.1-0.5 μL of T4 DNA ligase, and the rest is water; or The reaction system for constructing the library includes a blunt-end ligation library construction reaction system and a linker reaction system, wherein: the blunt-end ligation library construction reaction system includes 4-7 μL of PCR product, 0.8-1.2 μL of 10×T4 DNA ligase buffer, 0.5-1.5 μL of 10 mM ATP, 0.1-0.5 μL of T4 polynucleotide kinase, 0.1-0.5 μL of T4 DNA ligase, 0.05-0.15 μL of 10 mM dNTP, and the rest is water; the product corresponding to the blunt-end ligation library construction reaction system is the first PCR product; The linker reaction system, in a volume of 10 μL, includes 4-7 μL of the first PCR product, 1.5-3 μL of the barcode linker, 0.8-1.2 μL of 10×T4 DNA ligase buffer, 0.1-0.5 μL of T4 polynucleotide kinase, 0.1-0.5 μL of T4 DNA ligase, and the rest is water.

8. Use of the primer combination according to claim 1 or 2 in preparing a kit for detecting NOTCH2NLC gene; Preferably, the kit is a kit as described in any one of claims 3-7.

9. A system for detecting NOTCH2NLC gene, characterized in that: The system comprises: The primary screening module receives the capillary electrophoresis results of the long-range amplification product and the capillary electrophoresis results of the repeated amplification product, calculates the number of GGC repeats based on the capillary electrophoresis results of the long-range amplification product, determines the positive and negative based on the capillary electrophoresis results of the repeated amplification product, and determines the flow direction based on the following analysis results: 1) When the capillary electrophoresis results of the long-range amplification product show the presence of three or more signal peaks with different GGC repeat numbers, the sequencing module is entered for third-generation long-read sequencing; 2) When the number of GGC repeats is higher than the threshold, the sequencing module is used for third-generation long-read sequencing; 3) When the capillary electrophoresis result of the long-range amplification product shows only one signal peak with a normal range of GGC repeat number, but the capillary electrophoresis result of the repeat amplification product is positive, the sequencing module is entered for third-generation long-read sequencing; 4) When the capillary electrophoresis result of the long-range amplification product shows two or fewer signal peaks with different GGC repeat numbers, the capillary electrophoresis result of the repeat amplification product is negative, and the GGC repeat number is less than the threshold, the result is directly output by the result output module; Sequencing module, which performs third-generation long-read sequencing based on the DNA library constructed from the long-range amplification products; and A result output module, which outputs the analysis result of the primary screening module or the sequencing result of the sequencing module; Wherein, the long-range amplification product includes a first long-range amplification product and / or a second long-range amplification product, the first long-range amplification product is obtained based on the first group of long-range amplification primer combinations in the primer combination as described in claim 1 or 2; the repeated amplification product is obtained based on the repeated amplification primer combination in the primer combination as described in claim 1 or 2; the second long-range amplification product is obtained based on the second group of long-range amplification primer combinations in the primer combination as described in claim 1 or 2.

10. The system according to claim 9, characterized in that The system meets one or more of the following conditions: (1) The first long-range amplification product, the second long-range amplification product, the repeated amplification product and / or the DNA library are obtained by using the kit according to any one of claims 3 to 7; (2) the first long-range amplification product and / or the second long-range amplification product are obtained by the following amplification procedure: pre-denaturation at 98°C for 10 minutes; denaturation at 98°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 4 minutes, 32 cycles; final extension at 72°C for 10 minutes; (3) The repeated amplification product was obtained by the following amplification procedure: pre-denaturation at 98°C for 10 minutes; denaturation at 98°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 68°C for 4 minutes, 35 cycles; final extension at 68°C for 10 minutes; (4) The GGC repeat number is calculated by the following formula: (fragment length - 188 bp) / 3X, where "X" is the capillary instrument coefficient; (5) The positive or negative result is determined based on the capillary electrophoresis results of the repeated amplification products. When the signal shows a continuous step-like signal and the signal peak extends beyond 280 on the horizontal axis, or the number of signal peaks exceeds 38, it is judged as a positive result; otherwise, it is judged as a negative result; (6) The threshold of the number of GGC repeats is 65; (7) The normal range of the GGC repeat number is ≤38.

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