CN114317713A - Gene diagnosis kit for Alzheimer's disease and application thereof - Google Patents

Abstract

The invention discloses a gene diagnosis kit for Alzheimer's disease and application thereof, wherein the kit comprises: primer sets for amplifying CDS regions of the following genes: APP, APOE, PSEN1, PSEN2, ADAM10, PLD3, APBB2, HFE, NOS3, PAXIP1, PLAU, SORL1, A2M, BLMH, MPO, ACE, and MAPT; the primer group comprises a multiplex PCR primer group and a bridge PCR primer group; the bridge PCR primer group comprises an I5index primer group and an I7index primer group, and the nucleotide sequence is shown in SEQ ID NO.1-NO. 150. The SNV/Indel of the target gene can be detected by combining multiplex PCR with a second-generation sequencing method.

Description

Gene diagnosis kit for Alzheimer's disease and application thereof

Technical Field

The invention relates to the technical field of molecular biology analysis and detection, and relates to a gene diagnosis kit for Alzheimer's disease and application thereof.

Background

Alzheimer's Disease (AD) is a degenerative Disease of the central nervous system, and the major neuropathological changes include extracellular senile plaques formed by deposition of β amyloid a β, neurofibrillary tangles in neurons caused by hyperphosphorylation of microtubule-associated protein tau, neuronal loss, amyloid angiopathy, and the like. According to the age of onset, AD can be classified into early-onset AD (EOAD, the age of onset is less than 65 years) and late-onset AD (LOAD, the age of onset is more than or equal to 65 years), EOAD presents familial aggregation, and LOAD is mainly sporadic. APP, PSENl, PSEN2 are the causative genes of familial EOAD and are also genetic susceptibility factors of sporadic LOAD. APP and its modified genes, PSEN1, PSEN2, ADAM10 gene variation can influence the AD risk by regulating secretase activity; the genetic variation of cholesterol metabolism regulating gene such as sortilin-related receptor 1(SORL1), alpha 2-macroglobulin coding gene A2M, tau protein coding gene MAPT and the like are found to increase the risk of AD. AD is a heterogeneous disease with multiple etiologies, the pathogenesis of which is very complex and may be the result of the interaction of multiple factors (including genetic factors, environmental factors, neurotransmitter function, immune function, etc.). The current major pathogenesis hypothesis includes: genetic factors, neuronal apoptosis hypothesis centered on the a β cascade, neurofibrillary tangle hypothesis mediated by abnormal phosphorylation of Tau protein, aging and oxidative stress, insulin resistance, metal homeostasis, neurotransmitter channel hypothesis, and the like. AD is a continuously developing disease that may be advantageously treated more early and comprehensively by monitoring its pathophysiological characteristics. The International Working Group (IWG) -1 diagnostic standard issued in 2007 incorporated biomarkers into AD diagnostic standards for the first time. The IWG-2 diagnostic standard issued 2014 classified AD-related biomarkers. The american national institute of aging and alzheimer's disease association use biomarkers as diagnostic criteria for preclinical AD and as supportive evidence for Mild Cognitive Impairment (MCI) and dementia of AD origin. Therefore, the attention on the diseases related to the vasculopathy and the corresponding genes plays an important role in the occurrence of the AD, the early detection of the AD is facilitated, and the important significance is realized on the risk factor screening, cognitive assessment, imaging examination and early management intervention of the AD.

Because the disease is slow in onset or hidden, patients and families often say that the disease starts too late to determine the onset time in time. Currently, means for detecting AD are mainly focused on neuropsychological tests, blood tests, neuroimaging tests, electroencephalograms, cerebrospinal fluid biomarker detection, and the like. The imaging method has the defects of high price, radioactive damage to a patient and the like, and the cerebrospinal fluid marker detection has the defects of large sample collection invasiveness, high technical requirement and the like, and can only detect AD at the onset sign or the onset initial stage.

Therefore, it is necessary to develop a gene diagnostic kit for alzheimer's disease with high throughput and high accuracy.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a gene diagnosis kit for Alzheimer's disease and application thereof, and the kit has high flux and high accuracy.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of embodiments of the present invention, there is provided a genetic diagnosis kit for alzheimer's disease, the kit comprising:

primer sets for amplifying CDS regions of the following genes: APP, APOE, PSEN1, PSEN2, ADAM10, PLD3, APBB2, HFE, NOS3, PAXIP1, PLAU, SORL1, A2M, BLMH, MPO, ACE, and MAPT;

the primer set for amplifying the CDS region of the following gene comprises a multiplex PCR primer set and a bridge PCR primer set;

the bridge PCR primer group comprises an I5index primer group and an I7index primer group, and the nucleotide sequence of the I5index primer group is shown in SEQ ID NO.1-NO. 40; the nucleotide sequence of the I7index primer group is shown in SEQ ID NO.41-NO. 150.

The multiplex PCR primer set comprises KSGene-A Mix and KSGene-B Mix, wherein the nucleotide sequence of the KSGene-A Mix is shown as SEQ ID NO.151-NO. 578; the nucleotide sequence of the KSGene-B Mix is shown as SEQ ID NO.579-NO. 1006.

Further, the kit further comprises an enzyme.

Further, the amplification system of the multiplex PCR primer set includes KSGene-A Mix: 8. mu.L (or KSGene-B)Mix: 8. mu.L), enzyme HT: 10. mu.L, genomic DNA, loading 20-200ng (volume calculated from actual concentration), dd H₂O to 30. mu.L, and the multiplex PCR primer set comprising either KSGene-A Mix or KSGene-B Mix for amplification respectively.

Furthermore, the amplification system of the bridge PCR primer group comprises 10 mu L of enzyme HT, 1 mu L of primer of I5index primer group, 1 mu L of primer of I7index primer group, and dd H₂O:18μL。

In a second aspect of the embodiments of the present invention, there is provided a method of using the kit, the method comprising:

extracting and obtaining peripheral blood genome DNA;

performing multiple PCR reaction on the peripheral blood genome DNA by using multiple PCR primer groups, and then purifying to obtain a target region PCR product;

carrying out bridge PCR on the target region PCR product by using a bridge PCR primer group, and then purifying to obtain a library;

mixing the libraries and then sequencing on a computer to obtain a sequencing result;

and comparing the sequencing result with the CDS region base of each gene to judge whether mutation exists.

In a third aspect of the embodiments of the present invention, an application of the kit in detecting an alzheimer's disease risk gene locus is provided.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides a gene diagnosis kit for Alzheimer's disease and application thereof, wherein a second-generation sequencing method is adopted to detect 17 gene mutations related to Alzheimer's disease, the related genes have important value in the development of AD from the aspects of prediction, development, auxiliary diagnosis and the like, the panel design takes human hg19 genome as a template, multiple CDS regions of 17 genes (APP, APOE, PSEN1, PSEN2, ADAM10, PLD3, APBB2, HFE, NOS3, PAXIP1, PLAU, SORL1, A2M, BLMH, MPO, ACE and MAPT) are captured, and the SNV/Indel of a target gene can be detected by adopting PCR combined with the second-generation sequencing method. The primers panel is divided into two groups A/B, and after optimization panel, by using human genome DNA standard as a template, more than 97% of target sites or sequences can be captured and amplified, and more than 95% of amplified fragments cover more than 10% of the average sequencing depth. High flux and high accuracy (> 99%).

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a generation of sequencing validation data for sample S1;

FIG. 2 is a generation of sequencing validation data for sample S2;

FIG. 3 is a generation of sequencing validation data for sample S3;

FIG. 4 is a generation of sequencing validation data for sample S4;

FIG. 5 is a generation of sequencing validation data for sample S5;

FIG. 6 is a generation of sequencing validation data for sample S6;

FIG. 7 is a generation of sequencing validation data for sample S7;

FIG. 8 is a generation of sequencing validation data for sample S8;

FIG. 9 is a generation of sequencing validation data for sample S9;

FIG. 10 is a generation of sequencing validation data for sample S10.

Detailed Description

The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.

The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.

The technical scheme of the invention has the following general idea:

detecting 17 gene mutations related to the Alzheimer disease by adopting a second-generation sequencing method: APP, APOE, PSEN1, PSEN2, ADAM10, PLD3, APBB2, HFE, NOS3, PAXIP1, PLAU, SORL1, A2M, BLMH, MPO, ACE, MAPT.

Extracting peripheral blood genome DNA, capturing CDS regions of 17 genes by adopting a multiplex PCR method, carrying out bridge PCR on a prepared DNA library on a flow cell, simultaneously carrying out sequencing while synthesizing each basic group of a library DNA fragment, carrying out single-end (single-read) or double-end (pair-end) sequencing under the condition of ensuring efficiency and accuracy, and realizing direct and real-time detection; a large number of samples can be mixed in one system through different indexes, data with true central flux and high coverage are generated, and meanwhile, analysis and detection of gene hot spot regions can be carried out.

The detection result is applied to the aspects of predicting the occurrence risk of diseases, disease progression, auxiliary diagnosis and the like.

The terms to which the invention relates:

(1) adapter, a known short nucleotide sequence, is used to link unknown target sequencing fragments

(2) index or barcode, an oligonucleotide chain consisting of several bases, for distinguishing different samples in mixed sequencing

(3) insert, the target sequence to be sequenced, is located between two adapters.

Specifically, the primer set comprises a multiplex PCR primer set and a bridge PCR primer set;

the nucleotide sequence of the multiplex PCR primer group is shown as SEQ ID NO.151-NO. 1006;

the nucleotide sequence of the bridge PCR primer group is shown as SEQ ID NO.1-NO. 150;

the 3' end of the primers in the bridge PCR primer set is provided with oligo-dT, the length is 6-40 bases (namely nucleotides, in the invention, the base refers to the nucleotide, and the nucleotide is used interchangeably), and the preferable length is 18-24 bases;

the 5 'end of the primers in the bridge PCR primer set comprises sequences compatible with the 5' linker sequence (Adapter) of the sequencing library. Only DNA molecules with linkers added to both ends are able to undergo sequencing reactions by synthesis, as determined by the results of the linkers themselves.

On the one hand, it is also a difficulty of the present invention to select suitable primers panel so that more than 97% of the target site or sequence can be captured and amplified.

The invention divides the primer panel (namely the multiplex PCR primer group) into two groups A/B, and the optimized panel takes the human genome DNA standard as a template, more than 97 percent of target sites or sequences can be captured and amplified, and more than 95 percent of amplified fragments cover more than 10 percent of the average sequencing depth.

On the other hand, because the sequencing capacity of the sequencing instrument is far greater than the sequence quantity of a test sample, in order to avoid instrument waste, the simultaneous determination of a plurality of samples by one lane becomes a natural idea. However, in order to distinguish the sequences of multiple samples, it is necessary to add specific "tags" to the different samples, which are the barcodes, so that the data of the different samples can be separated in the subsequent data analysis. Briefly, barcode is an "identity card" that is used to distinguish between different samples in a mixed sample during sequencing. The primers in the bridge PCR primer group contain a tag barcode; there are two principles for the selection of barcode: base balancing and laser balancing, and selecting the proper barcode is also one of the difficulties of the present invention. If the base composition of the barcode combination is not balanced, which may lead to sequencing to the bases, software processing of the sequencing signal is obstructed, and the bases cannot be accurately identified (base-calling), which is shown by a reduced QV value and a fluctuating% Q30 curve. The barcode combination of the invention has good balance and high QV value.

In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.

Hereinafter, a gene diagnosis kit for alzheimer's disease and its application will be described in detail with reference to examples, comparative examples and experimental data.

Example 1 Gene diagnostic kit for Alzheimer's disease and method of use

Gene diagnosis kit for Alzheimer's disease

The kit comprises:

1. multiplex PCR System (primer sets in Table 1)

KSGene-A Mix: the nucleotide sequence is shown as SEQ ID NO.151-NO. 578; comprises an upper primer (SEQ ID NO.151-NO.578) and a lower primer (SEQ ID NO.579-NO.1006), and the upper primer and the lower primer correspond to each other in sequence one by one, for example, the upper primer of SEQ ID NO.151 corresponds to the lower primer of SEQ ID NO. 365;

KSGene-B Mix: the nucleotide sequence is shown as SEQ ID NO.579-NO. 1006; comprises an upper primer (SEQ ID NO.579-NO.792) and a lower primer (SEQ ID NO.793-NO. 1006); and the upper primer and the lower primer are in one-to-one correspondence in sequence, for example, the upper primer of SEQ ID NO.579 corresponds to the lower primer of SEQ ID NO. 793;

KSGene-A Mix；

EnzymeHT；

dd H₂O。

the KSGene-A Mix and the KSGene-B Mix were separated into A, B tubes for PCR.

TABLE 1 KSGene-AMix primer set

TABLE 2 KSGene-B Mix primer set

2. Bridge PCR system (primer sets in tables 3 and 4)

I5index primer set: the nucleotide sequence is shown in SEQ ID NO.1-NO. 40;

i7index primer set: the nucleotide sequence is shown as SEQ ID NO.41-NO. 150;

EnzymeHT；dd H₂O。

TABLE 3-I5 index primer set

TABLE 4-I7 index primer set

Method for using gene diagnosis kit for Alzheimer's disease

Step S1, extracting and obtaining peripheral blood genome DNA;

the method specifically comprises the following steps: extracting peripheral blood genome DNA, and accurately quantifying the genome by using the Qubit.

S2, performing multiple PCR reaction on the peripheral blood genome DNA by the multiple PCR primer group, and then purifying to obtain a target region PCR product;

the step S2 is intended for target region amplification, and the step S2 specifically includes:

(1) adopting a 0.2ml PCR tube/96-hole PCR plate, and respectively configuring the following two PCR reaction systems in a clean bench:

TABLE 5 Tube A PCR Reaction

TABLE 6-Tube B PCR Reaction

TABLE 7 PCR amplification procedure (temperature of the heat cover set at 102-

If the DNA input is less than 10ng, the increase is suitably 1-2 cycles.

In the Table 5, the KSGene-A Mix was prepared by adding all primers shown in SEQ ID NO.151-NO. 578;

in Table 6, the second PCR primer set was added with all primers shown in SEQ ID Nos. 579-1006.

(2) PCR product purification

Respectively taking 25ul of A/B products and mixing the products in equal amount; 0.5 times volume of AMPure XP Beads (25 muL of magnetic Beads in a 50 muL system) is added into 50ul of the mixed solution, and the mixed solution is blown up and down by a pipette, so that the amplified product and the magnetic Beads are fully and uniformly mixed. The mixture was allowed to stand at room temperature for 2 minutes.

The magnetic beads are attracted by a strong magnet or magnetic stand until the solution is clear.

The supernatant was carefully pipetted and transferred to a new centrifuge tube, carefully avoiding washing onto the beads. The magnetic beads of this step can be discarded.

And adding 0.6 time of AMPure XP Beads of an original system (30 mu L of magnetic Beads are added in a 50 mu L system) into the supernatant, and blowing up and down by using a pipette to fully and uniformly mix the amplified product and the magnetic Beads. The mixture was allowed to stand at room temperature for 2 minutes.

The magnetic beads are attracted by a strong magnet or magnetic stand until the solution is clear. Carefully aspirate the supernatant with a pipette, discard the supernatant and retain the beads.

The beads were suspended by adding 100. mu.L of WASH11 and allowed to stand at room temperature for 2 minutes. The magnetic beads are attracted by a strong magnet or magnetic stand until the solution is clear. The supernatant was pipetted, discarded and the beads retained.

Add 100. mu.L of 80% ethanol and adsorb the beads on both sides of the magnetic frame for thorough bead washing.

The magnetic beads are attracted with a magnet or magnetic stand until the solution is clear. The supernatant was carefully removed with a pipette to avoid attracting to the beads.

Standing at room temperature until ethanol is completely volatilized. In the step, the magnetic beads can also be placed in a 50 ℃ oven for 5 minutes, and the ethanol is quickly evaporated to dryness. This step can never be omitted, otherwise the residual ethanol would seriously affect the yield and subsequent experiments.

S3, performing bridge PCR on the target region PCR product by using a bridge PCR primer set, and then purifying to obtain a library;

the step S3 specifically includes:

(1) adding the following PCR system into the Tube with the magnetic beads:

TABLE 8

Different samples were obtained using different barcode primers (I7 Index primer set) and the same Index was used for Tube A/B; each of the weekly blood samples was used with a barcode primer;

the corresponding relation of the selected primers in the I5index primer group and the I7index primer group is as follows:

the nucleotide sequence of the I5index primer group is shown in SEQ ID NO.1-NO. 40; the nucleotide sequence of the I7index primer group is shown as SEQ ID NO.41-NO. 150;

i5 and I7 are combined, and the combination of I7 and I5in the same batch is not repeated. Not in a one-to-one correspondence. One I7 was used per exception of the weekly blood samples; the same I5index was used for each sample A/B tube.

Such as: the first sample, I7 is SEQ ID NO.41, I5 is SEQ ID NO. 1-40;

the second sample I7 is SEQ ID NO.42, and I5 is SEQ ID NO. 1-40;

the third sample, I7, was selected from SEQ ID NO.43 and I5 was selected from SEQ ID NO.1-40.

PCR amplification was performed according to the following procedure:

TABLE 9

(2) PCR product recovery

And adding 0.9-time volume of AMPure XP Beads (27 mu L in a 30 mu L system) into the PCR product, and blowing up and down by using a pipette to fully and uniformly mix the recovered product with magnetic Beads. The mixture was allowed to stand at room temperature for 2 minutes.

The magnetic beads are attracted by a strong magnet or magnetic stand until the solution is clear.

Carefully aspirate the supernatant with a pipette, discard the supernatant and retain the beads.

Add 100. mu.L of 80% ethanol, and repeatedly adsorb the beads back and forth on different sides with a magnetic rack to sufficiently suspend the beads for washing.

The magnetic beads are attracted with a magnet or magnetic stand until the solution is clear. The supernatant was carefully removed with a pipette to avoid attracting to the beads.

Standing at room temperature until ethanol is completely volatilized. The magnetic beads in the step can also be placed in a 50 ℃ oven for about 2-5 minutes, and the ethanol is quickly evaporated to dryness.

Add 20. mu.L of Elution Buffer, suspend the beads well, and let stand at room temperature for 2min to elute the DNA. The magnetic beads were adsorbed by a magnet, and the resulting supernatant DNA solution was transferred to a new 1.5/0.5/0.2mL centrifuge tube/96-well PCR tube. The Elution Buffer is 10mM Tris-HCl, pH 8.0-8.5, and TE can be used instead.

S4, mixing the libraries and then performing computer sequencing to obtain a sequencing result;

and step S5, comparing the sequencing result with CDS region base of each gene, and judging whether mutation exists.

Application example 1

The test was performed on 10 samples using the kit and method of example 1, and the test results are shown in table 10 and fig. 1-10.

Watch 10

As can be seen from Table 10 and FIGS. 1 to 10, samples S1 to S10 all had mutations, in which

Sample 1: detection of 1 missense variation of apolipoprotein E (APOE): c.388T>C (p.cys130arg) (heterozygous), which results in a genotype change to E3/E4 after mutation. Type E4 increases the risk of developing AD [1]Reference 1]

Nicolas,et al.Screening of dementia genes by whole-exome sequencing in early-onset Alzheimer disease:input and lessons.Eur J Hum Genet.2016 May；24(5):710-6.

Sample 2: missense variation of HFE gene detected at 1 position c.187C>G (p.his63asp) (heterozygous), the HGMD database included the site of this mutation as the pathological site of hemochromatosis (CM 960827). The mutation is shown in literature to be a risk factor related to AD, and can accelerate iron deposition of the brain^[2](ii) a Reference documents: [2]Daniel Berlin,et al.Evaluation of HFE(hemochromatosis)mutations as genetic modifiers in sporadic AD and MCI.Neurobiol Aging.2004 Apr；25(4):465-74.

Sample 3: c.1253C > T (p.Ser418Phe) (heterozygosis) is detected as the missense mutation of the A2M gene at 1, and whether the mutation has pathological significance is not clarified at all according to database and literature search, but APOE and A2M genes are reported as risk factors of late-onset AD in documents. [3]

[3] Korean, Zhang Shenglin, etc., APOE, A2M, ACE gene and Han's Alzheimer disease. [J]2008,39(8), 692-.

Sample 4: missense variation detected by HFE gene Exon 2, c.187C > G (p.His63Asp) (heterozygous), and the HGMD database records that the variation site is the pathological mutation site of hemochromatosis (CM960827), and the literature indicates that the variation is an AD related risk factor and can accelerate the iron deposition of the brain;

the apolipoprotein E (APOE) has 1 missense variation, namely c.388T > C (p.Cys130Arg) (heterozygosis), and the genotype of the apolipoprotein E (APOE) is changed into the E3/E4 type after the missense variation. Type E4 is a high risk genotype for AD.

Sample 5: the sample detects 1 HFE missense variation, namely c.187C > G (p.His63Asp) (heterozygous), and the HGMD database records that the variation site is the pathological mutation site of hemochromatosis (CM 960827). There is literature suggesting that this variation is a risk factor associated with AD, possibly accelerating brain iron deposition; missense mutation of NOS3 gene, c.382C > T (p.Arg128Trp) (heterozygous), was not clarified at all whether it had pathological significance or not, based on database and literature search.

Sample 6: the apolipoprotein E (APOE) genotype was detected as E3/E4. Type E4 increases the risk of AD.

Sample 7: the sample detects 1 APP gene intron variation, c.2064+4C > T (heterozygous), is positioned near the splicing site, whether the mutation has pathological significance is not clear at all, and the clinical diagnosis MCI of a patient indicates the possible risk of the disease, but further examination is needed if the disease is still needed.

Sample 8: the sample detects 1 APBB2 missense mutation c.1781A > G (p.Gln594Arg) (heterozygous), and whether the mutation has pathological significance is not clarified at the moment according to database and literature search.

Sample 9: the sample detects 1 ACE gene deletion variant, c.1324delC (p.Arg442ValfsTer14) (hybrid), and whether the mutation has pathogenic significance is not determined at all, but the mutation can cause the amino acid coding to terminate early and influence the activity of serum ACE (angiotensin converting enzyme). There are reports in the literature that serum ACE activity may be a potential risk factor for the development of AD, the former being a potential biological marker for AD disease states, particularly moderate to severe AD [4 ].

[4] Study of the role of serum angiotensin converting enzyme activity in alzheimer's disease [ D ] Qingdao university, 2016.

Sample 10: the sample detects that 1A 2M gene has intron variation, c.2126-6_2126-2del (heterozygous), and whether the sample has pathological significance or not is not determined temporarily according to database and literature search; the subject was diagnosed with MCI and had a suspicious family history, and APOE, A2M genes have been reported in the literature as risk factors for late-onset AD. But further examination is required to determine if the disease is present.

According to the detection result, whether the detected gene is normal or not can be judged, if the risk gene of the detected person has variation, the possibility of suffering from the disease is suggested, but whether the person suffers from the disease needs to be further checked, because the Alzheimer disease is influenced by various factors, the prevention of the Alzheimer disease can be improved. If there are no genetic abnormalities in it, then there is essentially no need for specialized targeted health management.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.

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<213> Artificial Sequence (Artificial Sequence)

<400> 19

aatgatacgg cgaccaccga gatctacacc gtaagccaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 20

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aatgatacgg cgaccaccga gatctacaca cgttaagaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 21

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aatgatacgg cgaccaccga gatctacaca ggcgaagaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 22

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aatgatacgg cgaccaccga gatctacact aatcttaaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 23

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

aatgatacgg cgaccaccga gatctacacc aggacgtaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 24

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

aatgatacgg cgaccaccga gatctacacg tactgacaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 25

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

aatgatacgg cgaccaccga gatctacaca ttactcgaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 26

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

aatgatacgg cgaccaccga gatctacact ccggagaaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 27

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aatgatacgg cgaccaccga gatctacacc gctcattaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 28

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

aatgatacgg cgaccaccga gatctacacg agattaaaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 29

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

aatgatacgg cgaccaccga gatctacaca ttcagccaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 30

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

aatgatacgg cgaccaccga gatctacacg aattcgtaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 31

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aatgatacgg cgaccaccga gatctacacc tgaagctaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 32

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aatgatacgg cgaccaccga gatctacact aatgcgcaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 33

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

aatgatacgg cgaccaccga gatctacacc ggctaaaaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 34

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

aatgatacgg cgaccaccga gatctacact ccccgtgaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 35

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

aatgatacgg cgaccaccga gatctacact ctggcgcaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 36

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

aatgatacgg cgaccaccga gatctacaca gcgatagaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 37

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

aatgatacgg cgaccaccga gatctacacc atcgtttaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 38

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

aatgatacgg cgaccaccga gatctacact gagaccaaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 39

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aatgatacgg cgaccaccga gatctacaca cgtcctgaca ctctttccct acacgacgct 60

cttccgatc 69

<210> 40

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

aatgatacgg cgaccaccga gatctacacg tcagtacaca ctctttccct acacgacgct 60

cttccgatc 69

Claims (6)

1. A gene diagnosis kit for Alzheimer's disease, comprising:

primer sets for amplifying CDS regions of the following genes: APP, APOE, PSEN1, PSEN2, ADAM10, PLD3, APBB2, HFE, NOS3, PAXIP1, PLAU, SORL1, A2M, BLMH, MPO, ACE, and MAPT;

the primer set for amplifying the CDS region of the following gene comprises a multiplex PCR primer set and a bridge PCR primer set;

the bridge PCR primer group comprises an I5index primer group and an I7index primer group, and the nucleotide sequence of the I5index primer group is shown as SEQ ID NO.1-NO. 40; the nucleotide sequence of the I7index primer group is shown in SEQ ID NO.41-NO. 150.

The multiplex PCR primer set comprises KSGene-A Mix and KSGene-B Mix, wherein the nucleotide sequence of the KSGene-A Mix is shown as SEQ ID NO.151-NO. 578; the nucleotide sequence of the KSGene-B Mix is shown as SEQ ID NO.579-NO. 1006.

2. The kit for gene diagnosis of alzheimer's disease as claimed in claim 1, wherein said kit further comprises enzyme ht enzyme.

3. The kit for gene diagnosis of Alzheimer's disease according to claim 1, wherein the amplification system of the multiplex PCR primer set comprises KSGene-A Mix: 8. mu.L (or KSGene-B Mix: 8. mu.L), Enzyme HT: 10. mu.L, genomic DNA, loading amount of 20-200ng (volume calculated based on actual concentration), dd H₂O complement to 30. mu.L, KSGene-A Mix and KSGene-B Mix, amplified in two tubes A \ B.

4. The kit for genetic diagnosis of Alzheimer's disease according to claim 1, wherein the amplification system of the bridge PCR primer set comprises EnzymeHT 10. mu.L, primer 1. mu.L of I5index primer set, primer 1. mu.L of I7index primer set, dd H₂O:18μL。

5. A method of using the kit of any one of claims 1 to 4, wherein the method comprises:

extracting and obtaining peripheral blood genome DNA;

performing multiple PCR reaction on the peripheral blood genome DNA by using multiple PCR primer groups, and then purifying to obtain a target region PCR product;

carrying out bridge PCR on the target region PCR product by using a bridge PCR primer group, and then purifying to obtain a library;

mixing the libraries and then sequencing on a computer to obtain a sequencing result;

and comparing the sequencing result with the CDS region base of each gene to judge whether mutation exists.

6. Use of the kit of claims 1-4 for detecting a genetic locus at risk of alzheimer's disease.

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