CN112831558B

CN112831558B - Early screening method and kit for Crohn disease susceptibility genes

Info

Publication number: CN112831558B
Application number: CN202110261808.5A
Authority: CN
Inventors: 徐力
Original assignee: Beijing Kelidandi Biomedical Technology Co ltd
Current assignee: Beijing Kelidandi Biomedical Technology Co ltd
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2022-12-20
Anticipated expiration: 2041-03-10
Also published as: CN112831558A

Abstract

The invention provides an early screening method and a kit of Crohn's disease susceptibility genes, and particularly relates to a method and a kit for screening out a combination of SNP (single nucleotide polymorphism) sites of the Crohn's disease susceptibility related genes by considering the difference of gene profiles of diseased people and healthy people, and carrying out wide (high-throughput detection sites and high-throughput detection samples) screening and detection on genetic markers related to the Crohn's disease by using a nucleic acid mass spectrometer. The method has high detection success rate, good technical reproducibility and high cost performance, can realize the detection of multiple genes of a single small sample, and meets the maximum use of the small sample; the method has the technical advantages of high accuracy and high sensitivity, the detection result is stable, and the detection positive rate is improved.

Description

Early screening method and kit for Crohn disease susceptibility genes

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an early screening method and a kit for a Crohn disease susceptibility gene.

Background

Crohn's Disease (CD) is an inflammatory disease of the intestinal tract of unknown origin, also known as Crohn's disease, and regional enteritis, and can occur in any part of the gastrointestinal tract, and may have a certain relationship with infection, humoral immunity, and cellular immunity. Is an autoimmune abnormal disease which is generated by a human with susceptibility bodies (carrying susceptibility genes) under the stimulation of external environmental factors, and is a gastrointestinal chronic inflammatory granulomatous disease which is commonly participated in by a plurality of genes. A large amount of signal channel research evidences show that the related gene locus is closely related to the genetic susceptibility of the CD, plays an important role in the pathogenesis of the CD, and lays a theoretical foundation for the research of the future pathogenesis of the CD and the related gene screening treatment.

Single Nucleotide Polymorphisms (SNPs) are genetic markers, which refer to polymorphisms in DNA sequences at the genomic level due to single nucleotide variations. The occurrence frequency in the population is more than 1 percent, and the expression comprises the conversion and inversion of single bases, the insertion or deletion of the single bases and the like, is a new genetic marker, and can provide reliable and effective scientific basis for the prediction, diagnosis and treatment of diseases and the development of novel medicaments.

The roles that SNPs play in disease gene localization mainly include: 1. searching for pathogenic SNPs in a disease localization region, wherein the occurrence of the SNPs can directly cause changes on the transcription level and the translation level of genes, namely, the expression quantity of the genes or the composition structure of protein of gene products is changed, so that certain diseases are caused or individuals are susceptible to certain special environments; SNPs are closely linked to a disease or phenotype as a genetic marker. In recent years, the prediction of the occurrence and development of diseases by using SNP has become a hot spot of clinical and scientific researchers, and the application value of the SNP in the prediction of important diseases such as tumors, cardiovascular and cerebrovascular diseases and the like is very early seen.

The cause of Crohn's disease is unknown, and it may be related to infection, immunity and heredity. The disease mainly affects the small intestine and colon, causes proliferative diseases penetrating through various layers of the intestinal wall, invades mesentery and local lymph nodes, and has the about high incidence rate

The onset of Crohn's disease is usually more insidious, and it usually takes several months to years from the early symptoms to the accurate diagnosis. The course of disease is chronic, with active periods and remission periods of different lengths alternating, and there is a tendency of recurrence throughout the life. A few of the acute cases have acute onset and can be manifested as acute abdominal symptoms, which closely resemble acute appendicitis or acute ileus. The occurrence of the Crohn's disease may be accompanied by other inflammatory bowel diseases, the involved digestive tract positions are wide, and the clinical diagnosis can be carried out by combining blood examination, stool examination, small intestine CT, magnetic resonance examination, colonoscopy and small intestine scope examination, so that the complicated detection process causes great inconvenience to patients. Clinically, the disease can be divided into an early stage (symptoms are not obvious and are one of the important reasons for the difficulty in diagnosing the disease), an active stage (symptoms are obvious), a remission stage (symptoms are improved after effective treatment) or chronic activity (caused by dependence on or resistance to medicaments and irregular treatment). Pathologically, it can be divided into early stage, active stage, chronic inflammation stage or regressive stage, etc. The disease has different stages, large difference of clinical manifestations, no symptom in early stage, obvious manifestation in active stage and slight symptom in remission stage. The latest clinical research shows that the research on the Crohn's disease discovers the existence of one genetic marker for the first time, and can realize personalized medicine treatment on different patients according to the genetic marker. At present, no effective early detection method exists in clinic, so a high-sensitivity, economic and simple molecular genetic marker screening method needs to be established urgently, the early diagnosis and treatment are carried out in advance by screening susceptibility genes of the Crohn disease, the gospel is brought to patients with early Crohn disease, and the personalized treatment and better prognosis are brought to the patients with the Crohn disease.

Disclosure of Invention

The invention aims to provide a method and a kit for early screening of a Crohn disease susceptibility gene.

The invention provides an early screening kit of a Crohn's disease susceptibility gene, which comprises a PCR amplification primer pair group, wherein the PCR amplification primer pair group comprises a primer pair for specifically amplifying SNP loci selected from the following groups: rs72553867, rs4409764, rs3731772, rs17525495, rs13361189, rs9637876, rs10065172, rs2241880, rs6754677, rs2066844, rs2066847, rs7574865, rs1004819, rs1495465, rs11465788, rs4343, rs744166, and rs11235604.

In another preferred example, the kit is used for detecting SNP gene mutation sites of the Crohn's disease susceptibility gene based on multiple PCR flight time mass spectrometry.

In another preferred embodiment, the kit further comprises a single base extension primer set.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs72553867 is shown in SEQ ID No.1 to SEQ ID No. 2.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs4409764 is shown in SEQ ID No.3 to SEQ ID No. 4.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs3731772 is shown in SEQ ID No.5 to SEQ ID No. 6.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs17525495 is shown as SEQ ID No.7 to SEQ ID No. 8.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs13361189 is shown as SEQ ID No.9 to SEQ ID No. 10.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs9637876 is shown as SEQ ID No.11 to SEQ ID No. 12.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs10065172 is shown as SEQ ID No.13 to SEQ ID No. 14.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs2241880 is shown as SEQ ID No.15 to SEQ ID No. 16.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs6754677 is shown as SEQ ID No.17 to SEQ ID No. 18.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs2066844 is shown as SEQ ID No.19 to SEQ ID No. 20.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs2066847 is shown as SEQ ID No.21 to SEQ ID No. 22.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs7574865 is shown as SEQ ID No.23 to SEQ ID No. 24.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs1004819 is shown as SEQ ID No.25 to SEQ ID No. 26.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs1495465 is shown as SEQ ID No.27 to SEQ ID No. 28.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs11465788 is shown as SEQ ID No.29 to SEQ ID No. 30.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs4343 is shown as SEQ ID No.31 to SEQ ID No. 32.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs744166 is shown as SEQ ID No.33 to SEQ ID No. 34.

In another preferred example, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs11235604 is shown as SEQ ID No.35 to SEQ ID No. 36.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs72553867 is shown as SEQ ID NO. 37.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs4409764 is shown as SEQ ID NO. 38.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs3731772 is shown as SEQ ID NO. 39.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs17525495 is shown as SEQ ID No. 40.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs13361189 is shown as SEQ ID NO. 41.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs9637876 is shown as SEQ ID No. 42.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs10065172 is shown as SEQ ID No. 43.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs2241880 is shown as SEQ ID No. 44.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs6754677 is shown as SEQ ID NO. 45.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs2066844 is shown as SEQ ID NO. 46.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs2066847 is shown as SEQ ID No. 47.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs7574865 is shown as SEQ ID NO. 48.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs1004819 is shown as SEQ ID NO. 49.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs1495465 is shown as SEQ ID No. 50.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs11465788 is shown as SEQ ID NO. 51.

In another preferred example, in the single-base extension primer set, the extension primer for rs4343 is shown as SEQ ID NO. 52.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs744166 is shown as SEQ ID No. 53.

In another preferred embodiment, in the single-base extension primer set, the extension primer for rs11235604 is shown as SEQ ID No. 54.

In another preferred embodiment, the kit comprises a first container, and the PCR amplification primer pair group is contained in the first container.

In another preferred embodiment, the kit comprises a second container, and the single-base extension primer set is contained in the second container.

In another preferred embodiment, the kit comprises a third container, and the third container contains a PCR premix, and the PCR premix mainly comprises hot-start Taq enzyme, dNTPs, mgCl ₂ And PCR buffer solution.

In another preferred embodiment, the kit comprises a fourth container containing shrimp alkaline phosphatase (SAPEnzyme).

In another preferred embodiment, the kit comprises a fifth container, and the fifth container contains an SAP buffer solution.

In another preferred embodiment, the kit comprises a sixth container containing an elongase (iPLEX Enzyme) therein.

In another preferred embodiment, the kit comprises a seventh container comprising ddNTPs.

In another preferred embodiment, the kit comprises an eighth container containing an extension reaction buffer.

In another preferred example, the kit further comprises pure water.

In a second aspect of the invention, a method for detecting SNP gene mutation sites of Crohn's disease susceptibility genes based on multiple PCR flight time mass spectrometry is provided, which comprises the following steps:

(1) Carrying out PCR amplification by taking the peripheral blood genome DNA of a sample to be detected as a template to obtain an amplification product;

(2) SAP treatment of the amplification product of step (1) with shrimp alkaline phosphatase;

(3) Carrying out single base extension reaction on the purified product in the step (2) by using an extension primer to obtain an extension product;

(4) Purifying the extension product with desalting resin;

(5) And (5) detecting and analyzing by a mass spectrum platform, and judging whether genetic variation exists.

In another preferred example, in the step (1), during the PCR amplification, SNP sites selected from the following group are specifically amplified: rs72553867, rs4409764, rs3731772, rs17525495, rs13361189, rs9637876, rs10065172, rs2241880, rs6754677, rs2066844, rs2066847, rs7574865, rs1004819, rs1495465, rs11465788, rs4343, rs744166, rs11235604.

In another preferred example, in the step (1), during the PCR amplification, the amplification primer pair group is used for PCR amplification.

In another preferred example, in the step (3), the single-base extension reaction is performed using the single-base extension primer set.

The third aspect of the invention provides the application of the PCR amplification primer pair group for preparing a detection kit, wherein the detection kit is used for detecting SNP gene mutation sites of susceptibility genes of Crohn's disease;

the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 36.

The fourth aspect of the invention provides the use of a single base extension primer group for preparing a detection kit, wherein the detection kit is used for detecting SNP gene mutation sites of susceptibility genes of Crohn's disease;

the single-base extension primer group comprises extension primers with sequences shown as SEQ ID NO.37 to SEQ ID NO. 54.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The invention provides an early screening method and a kit for Crohn's disease susceptibility genes, which screen 18 SNP sites of the Crohn's disease susceptibility related genes by comparing the difference of gene profiles of healthy people and diseased people, and further can utilize a nucleic acid mass spectrometer to carry out wide (high-throughput detection sites and high-throughput detection samples) screening and detection on genetic markers related to the Crohn's disease. Through multi-round screening, a multiplex PCR amplification primer pair which can carry out high-efficiency multiplex amplification on the 18 SNP loci and is suitable for mass spectrometric detection of MassARRAY nucleic acid is obtained, and a suitable extension primer is obtained through screening, so that high-accuracy and high-sensitivity detection on the 18 SNP loci is realized, the detection result is stable, and the detection positive rate is improved.

The determination method of the application is used for detecting the SNP gene mutation sites of the Crohn disease susceptibility genes based on a multiplex PCR technology and a MassARRAY nucleic acid mass spectrum technology, and can simultaneously detect 18 sites.

Multiplex PCR (multiplex PCR), also called multiplex primer PCR or composite PCR, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments, and the reaction principle, reaction reagents and operation process are the same as those of ordinary PCR.

There are many factors that affect multiplex PCR reactions, such as:

(1) The imbalance of the reaction system causes some dominant primers and templates thereof to be rapidly amplified in the previous rounds of reactions, and a large amount of amplification products are obtained, and the amplification products are good inhibitors of DNA polymerase. Therefore, the polymerization ability of polymerase is more and more strongly inhibited with the occurrence of a large amount of amplification products, and thus, primers and templates thereof which are at a disadvantage in the early stage are more difficult to react, and finally, the amount of amplification products is so small that they cannot be detected.

(2) The primer specificity, if the primer has stronger binding force with other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, thereby leading to the reduction of the amplification efficiency.

(3) The optimal annealing temperatures are different, a plurality of pairs of primers are placed in a system for amplification, and the optimal annealing temperatures of each pair of primers are required to be close to each other because the annealing temperatures for PCR reaction are the same.

(4) Primer dimers, including dimers between primers and hairpin structures formed by the primers themselves, are third-party DNA-mediated dimers, and these dimers, like non-specific primers, interfere with the competition between primers and target binding sites, affecting amplification efficiency.

Although several factors affecting amplification efficiency are mentioned above, more are not clear. To date, there is no effective method for clearly predicting amplification efficiency.

Although the multiplex PCR-time-of-flight mass spectrometry detection technology can carry out ultrahigh-flux detection, the requirement on the quality of a PCR amplification product is high. The inventor finds in research that the existing amplification primer and extension primer capable of carrying out detection by a multiplex fluorescence PCR method are directly applied to multiplex PCR-time-of-flight mass spectrometry, and have many defects, such as false negative of mass spectrometry caused by incapability of carrying out single base extension reaction, low sensitivity and poor repeatability, which are difficult to meet clinical application. Therefore, the inventor redesigns a plurality of pairs of amplification primers and extension primers aiming at each detection site, performs multiple combined detection verification under the condition that single-site detection can meet the requirement, and finally obtains a multiple PCR detection system and extension primers which have high sensitivity, good specificity and stable detection result and are suitable for flight time mass spectrometry detection through a large amount of test screening.

The invention adopts a multiplex PCR method to amplify a target sequence, artificially designs a plurality of pairs of primers, optimally selects and verifies the primers, and finally determines the nucleic acid detection kit which contains the following amplification primers and is used for detecting 18 sites in total of SNP gene mutation sites of the Crohn disease susceptibility gene.

TABLE 1 amplification primers

Wherein, F is an upstream primer, and R is a downstream primer.

The extension primers are shown in table 2:

TABLE 2 extension primers

The primer sequences listed in tables 1 and 2 can be synthesized by conventional polynucleotide synthesis methods.

Besides the amplification primer and the extension primer, the invention also provides a kit for detecting the SNP gene mutation site of the Crohn disease susceptibility gene, and the specific contents of the components in the kit are as follows:

TABLE 3 kit Components

The invention also provides a method for detecting the SNP gene mutation site of the Crohn disease susceptibility gene based on the multiple PCR flight time mass spectrum, which comprises the following steps:

(4) Desalting resin to purify the extension product;

Further, in the step (1), during the PCR amplification process, SNP sites selected from the following group are specifically amplified: rs72553867, rs4409764, rs3731772, rs17525495, rs13361189, rs9637876, rs10065172, rs2241880, rs6754677, rs2066844, rs2066847, rs7574865, rs1004819, rs1495465, rs11465788, rs4343, rs744166, rs11235604.

Further, in the step (1), in the PCR amplification process, the amplification primer pair group is used for PCR amplification.

Further, in the step (3), a single base extension reaction is performed using the extension primer set.

Further, the amplification conditions of step (1) are as follows: 95 deg.C for 3min;95 ℃ at 15s,62 ℃ at 15s,72 ℃ at 1min,45 cycles; keeping at 72 deg.C for 5min.

Further, the SAP treatment conditions of step (2) are: 40min at 57 deg.C and 5min at 65 deg.C.

Further, the conditions of the extension reaction of step (3) are as follows: 95 ℃ for 30s;95 ℃ for 5s, (54.5 ℃ for 5s,72 ℃ for 5s,5 cycles), 35 cycles; keeping at 72 deg.C for 5min.

The main advantages of the invention are:

the nucleic acid mass spectrum early screening method of the Crohn disease susceptibility gene considers the difference of the Crohn disease gene spectrum of healthy people and sick people, the detected susceptibility gene is more advanced, and a plurality of SNP sites which are susceptible to the Crohn disease are brought into the susceptible gene, and the sites have high detection success rate, good technical reproducibility and high cost performance;

the detection technology provided by the invention has obvious price advantage, and overcomes the disadvantages of high price, long time consumption, complicated operation and the like of the traditional single-base detection. The sensitivity in the aspect of early screening detection of the susceptible gene of the Crohn disease is higher, the flux is larger, the detection of multiple genes of a single small sample can be realized, and the maximized use of the small sample is met.

The nucleic acid mass spectrometry method for detecting the Crohn disease susceptibility gene early screen based on the MassARRAY nucleic acid mass spectrometry technology has the technical advantages of high accuracy and high sensitivity, is stable in detection result, has obvious advantages compared with Sanger sequencing, and improves the detection positive rate.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Peitang, eds., beijing: scientific Press, 2002, USA, sambrook, et al, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1

Feasibility analysis of SNP site screening of human Crohn's disease susceptibility related gene

The inventor screens and evaluates the site of Crohn's disease occurrence verified in large-scale pathology control group clinical research by searching genome-wide association study (GWAS) at home and abroad of NCBI, selects 18 single nucleotide polymorphism sites which are obviously related to the risk of Crohn's disease, and are independent of each other and have no linkage disequilibrium, so that the site selection of the invention has representativeness, independence and risk value accumulation, and can be used for evaluating the risk of individual Crohn's disease.

The selected SNP sites are as follows:

rs72553867、rs4409764、rs3731772、rs17525495、rs13361189、rs9637876、rs10065172、rs2241880、rs6754677、rs2066844、rs2066847、rs7574865、rs1004819、rs1495465、rs11465788、rs4343、rs744166、rs11235604。

example 2 system verification

The system verification comprises accuracy, specificity, sensitivity, precision, inter-personnel comparison and the like.

An accuracy verification scheme: 20 sites were tested and compared to Sanger sequencing, the expected target was 95%.

Specificity verification scheme: included in the accuracy is the expected target of 95%.

Sensitive verification protocol: the positive sample of human genome DNA is taken as a template, and the content of the labeled sample DNA is 1 ng/muL, 5 ng/muL, 10 ng/muL, 50 ng/muL and 100 ng/muL respectively for sensitivity investigation.

Precision validation protocol (including intra-batch, inter-batch, personnel comparisons, not involving inter-instrument comparisons) expected target 95%.

Internal precision: the same batch was repeated 3 times for each sample and the intra-batch precision was compared.

Batch precision: the same operator examines the same samples in multiple batches and compares the batch-to-batch precision.

The personnel alignment: 2 operators tested the same samples and compared the differences in results between the individuals.

The specific test steps are as follows:

1. DNA extraction: according to the operation steps provided by the instruction of a blood DNA extraction kit (a commercially available kit for rapidly and efficiently extracting genomic DNA) independently developed by Nanxin medical treatment, the human peripheral blood genomic DNA is prepared, and 50 mu LddH ₂ Eluting with oxygen;

2. PCR procedure

(1) Samples were diluted to 20 ng/. Mu.L;

(2) The following table was used to prepare a PCR reaction system (hereinafter, a single sample amount, 40ng of sample DNA in total)

TABLE 4 PCR reaction System

Reagent	W1(μL)	W2(μL)
			Water, ddH ₂ O	0.8	0.8
10PCR Buffer with 20mM	0.5	0.5
			25mM MgCl ₂	0.4	0.4
25mM dNTP mixture	0.1	0.1
			25 mu M amplification primer mixture	1	1
5U/. Mu.L PCR Taq enzyme	0.2	0.2
			20ng/μL DNA	2	2
Total volume	5.00	5.00

(3) Sealing the membrane, mixing with vortex for 30 seconds, and centrifuging at 500g for 1 minute;

(4) Place the plate on a PCR instrument for the following thermal cycling:

95 ℃ for 3 minutes

45 cycles:

(95 ℃ for 15 seconds)

62 ℃ for 15 seconds

72 ℃ for 1 minute

5 minutes at 72 DEG C

Keeping the temperature at 4 DEG C

2. SAP flow scheme

(1) Taking out the PCR plate, and centrifuging for 3 minutes at 500 g;

(2) SAP reaction systems (individual sample amounts below) were formulated as follows;

TABLE 5 SAP reaction System

Reagent	Per well sample (μ L)	×2
			ddH ₂ O	1.53	3.06
SAP buffer	0.17	0.34
			SAP enzyme (1.7U/. Mu.L)	0.3	0.6
Total volume	2.00	4.00

(3) Adding 2 mu LSAP mixed solution into each hole;

(4) Sealing the membrane, mixing with vortex for 30 seconds, and centrifuging at 500g for 1 minute;

(5) The plate was placed on a PCR instrument for the following thermal cycling:

40 minutes at 57 DEG C

5 minutes at 65 DEG C

Keeping the temperature at 4 DEG C

3. EXT (Single base extension) protocol

(1) Taking out the PCR plate, and centrifuging for 3 minutes at 500 g;

(2) The following table was followed to formulate the EXT reaction system (individual sample amounts below);

TABLE 6 EXT reaction System

Reagent	W1(μL)	W2(μL)
			ddH ₂ O	0.62	0.62
iPLEX buffer solution	0.2	0.2
			ddNTP mixed liquor	0.2	0.2
Extension primer mixture	0.94	0.94
			iPLEX enzyme	0.04	0.04
Total volume	2.00	2.00

(3) Adding 2 mu L of iPLEX extension mixed solution;

(5) Place the plate on a PCR instrument for the following thermal cycling:

95℃30s

35 cycles:

(95℃、5s

5 cycles:

(54.5℃5s

72℃5s))

72℃5min

keeping the temperature at 4 DEG C

4. Resin desalination

Taking out the PCR plate, and centrifuging for 3 minutes at 500 g; spreading clean Resin (Resin) on the sample plate hole, and air-drying for at least 10 min; adding 10uL of water into each hole with the sample in the sample plate; plate closed, vortex 10 seconds, 500g centrifugation for 1 minute; slightly inverting the sample plate in a volley manner, placing the sample plate on the sample plate with the resin, and then inverting the sample plate together with the sample plate (the two quick plates cannot move horizontally in the process) to allow the resin to fall into the holes; taking down the sample plate, sealing the sample plate, and shaking up for 3 minutes with the rotator upside down; centrifuge at 2000g for 5 minutes.

5. Dispensing spotting

MALDI-TOF (matrix assisted laser Desorption ionization-time of flight) mass spectrometer was used to obtain a clustering plot (clear homopolymeric) of each site of the data.

And (3) test results: the accuracy verification results of 1 sample are shown in table 7.

TABLE 7 accuracy verification (comparison of Primary sequencing with MassARRAY results)

SNP_ID	First generation sequencing results	MassARRAY results
			rs72553867	CC	CC
rs4409764	TT	TT
			rs3731772	TT	TT
rs17525495	GG	GG
			rs13361189	TT	TT
rs9637876	CC	CC
			rs10065172	CC	CC
rs2241880	AA	AA
			rs6754677	GA	GA
rs2066844	CC	CC
			rs2066847	CC	CC
rs7574865	TT	TT
			rs1004819	GG	GG
rs1495465	CC	CC
			rs11465788	CC	CC
rs4343	GA	GA
			rs744166	AA	AA
rs11235604	CC	CC

The site rs1495465 of this sample is used as an example, and the precision verification results are shown in table 5.

TABLE 8 result of site precision verification of rs1495465

On the whole, all the sites of the method are clustered clearly, basically have no gray areas, and the false detection is possibly small. The accuracy (including sensitivity and specificity) and precision of each site detection of the present application were verified and are shown in table 9.

TABLE 9 verification results of accuracy, sensitivity, specificity

SNP_ID

Accuracy of

Sensitivity of the probe

Specificity of

Precision in batch

Inter-batch precision

Comparison of persons

rs72553867

100％

5ng/μL

100％

rs4409764

100％

5ng/μL

100％

rs3731772

100％

5ng/μL

100％

rs17525495

100％

5ng/μL

100％

rs13361189

100％

5ng/μL

100％

rs9637876

100％

5ng/μL

100％

rs10065172

100％

5ng/μL

100％

rs2241880

100％

5ng/μL

100％

rs6754677

100％

5ng/μL

100％

rs2066844

100％

5ng/μL

100％

rs2066847

100％

5ng/μL

100％

rs7574865

100％

5ng/μL

100％

rs1004819

100％

5ng/μL

100％

rs1495465

100％

5ng/μL

100％

rs11465788

100％

5ng/μL

100％

rs4343

100％

5ng/μL

100％

rs744166

100％

5ng/μL

100％

rs11235604

100％

5ng/μL

100％

In the above table, the accuracy of 100% indicates that all positive samples were correctly detected and consistent with Sanger sequencing results; the specificity of 100 percent indicates that no false positive result appears in the detected sample; the batch precision is 100%, which indicates that the repeated detection results of the same batch of samples can be kept consistent; the batch precision is 100 percent, which indicates that the detection results of the same operator for detecting the same sample in multiple batches can be kept consistent; the personnel comparison of 100 percent shows that the detection results of 2 operators detecting the same sample can be kept consistent.

In conclusion, the nucleic acid mass spectrum early screening method of the Crohn disease susceptibility gene provided by the invention considers the difference of the Crohn disease gene spectrum of Chinese and European and American populations, the detected susceptibility gene is more advanced, a plurality of SNP sites which are susceptible to the Crohn disease are included, and the sites have high detection success rate, good technical reproducibility and high cost performance.

Comparative example 1 screening of PCR amplification primer set and extension primer

Aiming at each site, the inventor designs several to ten pairs of amplification primers and extension primers, and then verifies and optimizes the amplification primers and the extension primers to finally establish a multiplex PCR amplification primer and extension primer combination which can be used for MassARRAY nucleic acid mass spectrometry technology detection.

In this comparative example, the site rs1004819 is used as an example, and an amplification primer and an extension primer having partially unsatisfactory effects are exemplified.

Control primer pair 1:

F-1：ACGTTGGATGTGACCTGCTTTATGCTGT(SEQ ID NO.:55)

R-1：ACGTTGGATGATTTCTCACCTCCTCCTG(SEQ ID NO.:56)

control primer pair 2:

F-2：ACGTTGGATGACTGACCTGCTTTATGCT(SEQ ID NO.:57)

R-2：ACGTTGGATGATTTCTCACCTCCTCCTG(SEQ ID NO.:56)

control primer pair 3:

F-3：ACGTTGGATGTGACCTGCTTTATGCTGT(SEQ ID NO.:55)

R-3：ACGTTGGATGCCTCTGAGTTCGGCTTGG(SEQ ID NO.:58)

control extension primer 1:

Y-1：TATGCTGTGATTCTTACT(SEQ ID NO.:59)

control extension primer 2:

Y-2：TTTATGCTGTGATTCTTACT(SEQ ID NO.:60)

the primer pair of the invention comprises: SEQ ID NO.25 and 26

The extension primer of the invention: SEQ ID NO.49

In the single screening experiment, different extension primers are used for single base extension after single PCR amplification, and then mass spectrum detection is carried out on extension products, and the single detection result shows that the control primer pair 2 and the control primer pair 3 can normally work when being detected in a single system, but positive results cannot be obtained in a multiple system.

In the multiplex system, the combination of the control primer pair 1 and the extension primer (SEQ ID NO. 49) of the invention has the detection sensitivity of 10 ng/mu L; the detection sensitivity of the combination of the control primer pair 1 and the control extension primer 1 is 50 ng/. Mu.L; the combination of the control primer pair 1 and the control extension primer 2 showed a detection sensitivity of 50 ng/. Mu.L. And the combination of the primer pairs shown in SEQ ID NO.25 and 26 and the extension primer shown in SEQ ID NO.49 can achieve the detection sensitivity of 5 ng/muL.

The results indicate that the control primer pair 2, 3 cannot effectively amplify the target nucleic acid sequence in the detection system and therefore cannot work in the detection system; control primer pair 1 can work in multiplex detection lines, but the sensitivity is poor. The combination of the primer pair (SEQ ID NO.25 and 26) and the extended primer (SEQ ID NO. 49) of the present invention can work normally in a multiple detection system, and the sensitivity is high and reaches 5 ng/. Mu.L.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Beijing Kelidandi biomedical science and technology Co., ltd

Early screening method and kit for <120> Crohn disease susceptibility genes

<130> P210098

<160> 60

<170> PatentIn version 3.5

<210> 1

<211> 26

<212> DNA

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<400> 1

acgttggatg tcatcagtgc ccttcg 26

<210> 2

<211> 26

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<400> 2

acgttggatg tgtcctcagc ggtttt 26

<210> 3

<211> 27

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<213> Artificial sequence

<400> 3

acgttggatg gaaaccaggc aaatcca 27

<210> 4

<211> 26

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<400> 4

acgttggatg ctaaagcagc cgcaaa 26

<210> 5

<211> 27

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<400> 5

acgttggatg gctcacttgt cccgtaa 27

<210> 6

<211> 27

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<400> 6

acgttggatg ggcttgtaga tggctgt 27

<210> 7

<211> 28

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<400> 7

acgttggatg gccaacgaac aggtatcc 28

<210> 8

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<400> 8

acgttggatg cccctcaaca tccctaaca 29

<210> 9

<211> 26

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<400> 9

acgttggatg gctggctcag ttcaca 26

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<400> 10

acgttggatg cacaggtctt ggcatta 27

<210> 11

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<400> 11

acgttggatg cagtgcccac agatacga 28

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<400> 12

acgttggatg tcacgaggtc aggagatt 28

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<400> 13

acgttggatg tcatcagtgc ccttcg 26

<210> 14

<211> 26

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<213> Artificial sequence

<400> 14

acgttggatg tgtcctcagc ggtttt 26

<210> 15

<211> 28

<212> DNA

<213> Artificial sequence

<400> 15

acgttggatg gaaggacagg ctatcaac 28

<210> 16

<211> 25

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<400> 16

acgttggatg tcaagcgtgg taggg 25

<210> 17

<211> 26

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<400> 17

acgttggatg tagggaaggc agaacg 26

<210> 18

<211> 27

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<213> Artificial sequence

<400> 18

acgttggatg gctaaacacc caacgac 27

<210> 19

<211> 28

<212> DNA

<213> Artificial sequence

<400> 19

acgttggatg tcggagggaa aggacagc 28

<210> 20

<211> 26

<212> DNA

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<400> 20

acgttgatgg gcgggatgga gtggaa 26

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<400> 21

acgttggatg tctaagggac aggtggg 27

<210> 22

<211> 29

<212> DNA

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<400> 22

acgttggatg tgtcagaatc agaagggat 29

<210> 23

<211> 26

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<400> 23

acgttggatg aatacggatg tctttg 26

<210> 24

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<400> 24

acgttggatg tcttgcttta ggagtt 26

<210> 25

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<400> 25

acgttggatg cataataaat gccagtttgc 30

<210> 26

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<400> 26

acgttggatg acctcctcct gtcaccc 27

<210> 27

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<400> 27

acgttggatg ggggtgcttt ggtag 25

<210> 28

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<400> 28

acgttggatg ctgggcaagg gtatt 25

<210> 29

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<400> 29

acgttggatg cagtattagg gtgggagtga 30

<210> 30

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<400> 30

acgttggatg caagatggct gaataggaac 30

<210> 31

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<400> 31

acgttggatg gccactccca tcctttc 27

<210> 32

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<400> 32

acgttggatg gggtttcaca gcacctctt 29

<210> 33

<211> 27

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acgttggatg tgtgatggga cttggtg 27

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acgttggatg tgatgtttct gcctgatt 28

<210> 35

<211> 27

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<400> 35

acgttggatg gagcggtgag ggagcag 27

<210> 36

<211> 27

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<400> 36

acgttggatg ctcaggacgg ggctgtg 27

<210> 37

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<400> 37

gggacctgcc tggca 15

<210> 38

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atctcaacac gataacaatg aca 23

<210> 39

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<400> 39

gtaattaaga taaaatgagg gca 23

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<400> 40

gtcgatagag aacctgagga g 21

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<400> 41

aagcagagtg tgcttgaaaa t 21

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<400> 42

aggctggagt gcaatgg 17

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<400> 43

ccacaaccct ggagaactac 20

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<211> 22

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<400> 44

cttaggagac gctctgtctc tt 22

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<400> 45

gctgatacat ggcaagtga 19

<210> 46

<211> 17

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<400> 46

ggcgggatgg agtggaa 17

<210> 47

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<400> 47

cagaagccct cctgcag 17

<210> 48

<211> 21

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<400> 48

aaaagttggt gaccaaaatg t 21

<210> 49

<211> 19

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<213> Artificial sequence

<400> 49

ttatgctgtg attcttact 19

<210> 50

<211> 23

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<400> 50

taagaacaaa ggcagtaaaa gaa 23

<210> 51

<211> 15

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<213> Artificial sequence

<400> 51

tgcctcgccc tgctt 15

<210> 52

<211> 18

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<400> 52

gacgaatgtg atggccac 18

<210> 53

<211> 27

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<400> 53

aactgtttgt tctataaatt actgtca 27

<210> 54

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<400> 54

agggccaagc aggcg 15

<210> 55

<211> 28

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<400> 55

acgttggatg tgacctgctt tatgctgt 28

<210> 56

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<400> 56

acgttggatg atttctcacc tcctcctg 28

<210> 57

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<400> 57

acgttggatg actgacctgc tttatgct 28

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<400> 58

acgttggatg cctctgagtt cggcttgg 28

<210> 59

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<400> 59

tatgctgtga ttcttact 18

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<211> 20

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<400> 60

tttatgctgt gattcttact 20

Claims

1. A kit for detecting SNP sites of Crohn's disease susceptibility genes is characterized by comprising a PCR amplification primer pair group, wherein the PCR amplification primer pair group comprises primer pairs for specifically amplifying the SNP sites of the following groups: rs72553867, rs4409764, rs3731772, rs17525495, rs13361189, rs9637876, rs10065172, rs2241880, rs6754677, rs2066844, rs2066847, rs7574865, rs1004819, rs1495465, rs11465788, rs4343, rs744166, and rs11235604; the kit further comprises a single-base extension primer group;

wherein, in the PCR amplification primer pair group:

the primer pair for specifically amplifying rs72553867 is shown in SEQ ID NO. 1-SEQ ID NO. 2;

the primer pair for specifically amplifying rs4409764 is shown in SEQ ID NO. 3-SEQ ID NO. 4;

the primer pair for specifically amplifying rs3731772 is shown as SEQ ID NO.5 to SEQ ID NO. 6;

the primer pair for specifically amplifying rs17525495 is shown in SEQ ID NO. 7-SEQ ID NO. 8;

the primer pair for specifically amplifying rs13361189 is shown as SEQ ID NO.9 to SEQ ID NO. 10;

the primer pair for specifically amplifying rs9637876 is shown as SEQ ID NO.11 to SEQ ID NO. 12;

the primer pair for specifically amplifying rs10065172 is shown as SEQ ID NO.13 to SEQ ID NO. 14;

the primer pair for specifically amplifying rs2241880 is shown as SEQ ID NO.15 to SEQ ID NO. 16;

the primer pair for specifically amplifying rs6754677 is shown as SEQ ID NO.17 to SEQ ID NO. 18;

the primer pair for specifically amplifying rs2066844 is shown as SEQ ID NO.19 to SEQ ID NO. 20;

the primer pair for specifically amplifying rs2066847 is shown in SEQ ID NO.21 to SEQ ID NO. 22;

the primer pair for specifically amplifying rs7574865 is shown as SEQ ID NO.23 to SEQ ID NO. 24;

the primer pair for specifically amplifying rs1004819 is shown as SEQ ID NO.25 to SEQ ID NO. 26;

the primer pair for specifically amplifying rs1495465 is shown in SEQ ID NO.27 to SEQ ID NO. 28;

the primer pair for specifically amplifying rs11465788 is shown as SEQ ID NO.29 to SEQ ID NO. 30;

the primer pair for specific amplification of rs4343 is shown as SEQ ID NO.31 to SEQ ID NO. 32;

the primer pair for specifically amplifying rs744166 is shown as SEQ ID NO.33 to SEQ ID NO. 34; the primer pair for specifically amplifying rs11235604 is shown as SEQ ID NO.35 to SEQ ID NO. 36;

in the single-base extension primer set:

the extension primer aiming at rs72553867 is shown as SEQ ID NO. 37;

the extension primer aiming at rs4409764 is shown as SEQ ID NO. 38;

the extension primer aiming at rs3731772 is shown as SEQ ID NO. 39;

the extension primer aiming at rs17525495 is shown as SEQ ID NO. 40;

the extension primer aiming at rs13361189 is shown as SEQ ID NO. 41;

the extension primer aiming at rs9637876 is shown as SEQ ID NO. 42;

the extension primer aiming at rs10065172 is shown as SEQ ID NO. 43;

the extension primer aiming at rs2241880 is shown as SEQ ID NO. 44;

the extension primer aiming at rs6754677 is shown as SEQ ID NO. 45;

the extension primer aiming at rs2066844 is shown as SEQ ID NO. 46;

the extension primer aiming at rs2066847 is shown as SEQ ID NO. 47;

the extension primer aiming at rs7574865 is shown as SEQ ID NO. 48;

the extension primer aiming at rs1004819 is shown as SEQ ID NO. 49;

the extension primer aiming at rs1495465 is shown as SEQ ID NO. 50;

the extension primer aiming at rs11465788 is shown as SEQ ID NO. 51;

the extension primer aiming at rs4343 is shown as SEQ ID NO. 52;

the extension primer aiming at rs744166 is shown as SEQ ID NO. 53;

the extension primer aiming at rs11235604 is shown as SEQ ID NO. 54.

2. The kit of claim 1, wherein the kit comprises a first container comprising the PCR amplification primer pair group.

3. The kit of claim 2, further comprising a second container containing the set of single-base extension primers.

4. The kit of claim 3, further comprising a third container containing a PCR premix comprising hot-start Taq enzyme, dNTPs, mgCl ₂ And PCR buffer.

5. The kit of claim 4, further comprising a fourth container, wherein the fourth container comprises shrimp alkaline phosphatase.

6. The kit of claim 5, further comprising a fifth container comprising an SAP buffer.

7. The kit of claim 6, further comprising a sixth container containing an elongase.

8. The kit of claim 7, further comprising a seventh container comprising ddNTPs.

The application of the PCR amplification primer group and the single base extension primer group is characterized in that the application is used for preparing a detection kit, and the detection kit is used for detecting SNP gene mutation sites of susceptibility genes of Crohn's disease;

the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 36;