CN112592972B

CN112592972B - Early screening method and kit for diffuse toxic goiter susceptibility genes

Info

Publication number: CN112592972B
Application number: CN202011584184.2A
Authority: CN
Inventors: 陈涛; 张静; 袁昕昕; 林金飞
Original assignee: Guangdong Longsee Medical Technology Co ltd
Current assignee: Guangdong Longsee Medical Technology Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2023-05-23
Anticipated expiration: 2040-12-28
Also published as: CN112592972A

Abstract

The invention provides an early screening method and a kit for diffuse toxic goiter susceptibility genes, and particularly relates to a method for screening out a combination of SNP loci of diffuse toxic goiter susceptibility genes of Chinese by taking the difference of gene spectrums of Chinese people and European people into consideration, and performing extensive (high-flux detection loci and high-flux detection samples) screening and inspection on genetic markers related to diffuse toxic goiter by using a nucleic acid mass spectrometer. The method has the advantages of high success rate of detection, good technical reproducibility and high cost performance, can realize the detection of multiple genes of a single small sample, and satisfies the maximum use of the small sample; the method has the technical advantages of high accuracy and high sensitivity, and has stable detection result and improved detection positive rate.

Description

Early screening method and kit for diffuse toxic goiter 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 diffuse toxic goiter susceptibility genes.

Background

Diffuse toxic goiter (GD for short) is an autoimmune disease, mainly affecting thyroid. It is the most common cause of hyperthyroidism and is present at all ages, especially in women of child bearing age. A great deal of epidemiological evidence suggests that genetic factors play an important role in the pathogenesis of GD.

Single nucleotide polymorphisms (single nucleotide polymorphism, SNPs) are genetic markers, which refer to polymorphisms in DNA sequences at the genomic level due to variation of single nucleotides. The occurrence frequency in the crowd is more than 1%, the expression of forms including single base conversion, inversion, single base insertion or deletion and the like is a new genetic marker, and a reliable and effective scientific basis can be provided for the prediction, diagnosis and treatment of diseases and the development of novel medicines.

The roles played by SNPs in disease gene localization mainly include: 1. searching for pathogenic SNPs in disease localized regions, the occurrence of which may directly lead to changes in gene transcription and translation levels, i.e., changes in gene expression levels or the composition of the gene product protein, thereby leading to the occurrence of a disease or rendering an individual susceptible to a particular environment; snp as a genetic marker, closely linked to disease or phenotype. In recent years, the prediction of occurrence and development of diseases by utilizing SNP has become a hotspot of clinical and scientific researchers, and the application value in the prediction of serious diseases such as tumor, cardiovascular and cerebrovascular diseases and the like has become the forefront.

Diagnosis can be determined clinically based on symptoms and signs of thyrotoxicosis in combination with results from laboratory tests (TSHs and free T4) and used as a basis for treatment evaluation. It is not uncommon for GD diagnostics to remain indeterminate even after all of these tests have been made. When this happens, TRAb test is needed, and the process is complicated and cannot be rapidly diagnosed. GD patients receiving ATD therapy at the same time have a higher recurrence rate after discontinuation of the drug, which is a major limitation of ATD therapy. It is therefore important to determine some predictive factors for a higher risk of relapse in GD patients, which may help to employ appropriate treatment methods for a given patient at the time of GD diagnosis. Clinical studies indicate that genetic susceptibility accounts for 79% of GD risk, while environmental factors account for 21%. At present, no effective early detection method exists clinically, so that a high-sensitivity, economical and simple molecular technology screening method needs to be established, and the domestic blank is filled.

Disclosure of Invention

The invention aims to provide an early screening method and a kit for diffuse toxic goiter susceptibility genes.

In a first aspect of the invention, there is provided an early screening kit for diffuse toxic goiter susceptibility genes, the kit comprising a PCR amplification primer set comprising a primer pair that specifically amplifies a SNP site selected from the group consisting of: rs2294025, rs7005834, rs2941522, rs907091, rs1453559, rs12150079, rs2872507, rs3087243, rs231775, rs229527, rs2284038, rs4969170, rs12101261, rs179243, rs40401, rs2069812 and rs2227306.

In another preferred embodiment, the kit detects SNP gene mutation sites of diffuse toxic goiter susceptibility genes based on multiplex PCR time-of-flight 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 rs2294025 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 rs7005834 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 rs2941522 is shown in SEQ ID NO.5 to SEQ ID NO. 6.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs907091 is shown in SEQ ID NO.7 to SEQ ID NO. 8.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs1453559 is shown in SEQ ID NO.9 to SEQ ID NO. 10.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs12150079 is shown in SEQ ID NO.11 to SEQ ID NO. 12.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs2872507 is shown in SEQ ID NO.13 to SEQ ID NO. 14.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs3087243 is shown in SEQ ID NO.15 to SEQ ID NO. 16.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs231775 is shown in SEQ ID NO.17 to SEQ ID NO. 18.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs229527 is shown in SEQ ID NO.19 to SEQ ID NO. 20.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs2284038 is shown in SEQ ID NO.21 to SEQ ID NO. 22.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs4969170 is shown in SEQ ID NO.23 to SEQ ID NO. 24.

In another preferred embodiment, in the PCR amplification primer pair group, the primer pair for specifically amplifying rs12101261 is shown in SEQ ID NO.25 to SEQ ID NO. 26.

In another preferred embodiment, in the PCR amplification primer pair set, the primer pair for specifically amplifying rs179243 is shown in SEQ ID NO.27 to SEQ ID NO. 28.

In another preferred embodiment, in the PCR amplification primer pair set, the primer pair for specifically amplifying rs40401 is shown in SEQ ID NO.29 to SEQ ID NO. 30.

In another preferred embodiment, in the PCR amplification primer pair set, the primer pair for specifically amplifying rs2069812 is shown in SEQ ID NO.31 to SEQ ID NO. 32.

In another preferred embodiment, in the PCR amplification primer pair set, the primer pair for specifically amplifying rs2227306 is shown in SEQ ID NO.33 to SEQ ID NO. 34.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs2294025 is shown in SEQ ID NO. 35.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs7005834 is set forth in SEQ ID NO. 36.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs2941522 is shown in SEQ ID NO. 37.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs907091 is set forth in SEQ ID NO. 38.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs1453559 is set forth in SEQ ID NO. 39.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs12150079 is set forth in SEQ ID NO. 40.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs2872507 is shown in SEQ ID NO. 41.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs3087243 is set forth in SEQ ID NO. 42.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs231775 is set forth in SEQ ID NO. 43.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs229527 is set forth in SEQ ID NO. 44.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs2284038 is set forth in SEQ ID NO. 45.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs4969170 is set forth in SEQ ID NO. 46.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs12101261 is shown in SEQ ID NO. 47.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs179243 is shown in SEQ ID No. 48.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs40401 is shown in SEQ ID NO. 49.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs2069812 is as shown in SEQ ID NO. 50.

In another preferred embodiment, in the single base extension primer set, the extension primer for rs2227306 is shown in SEQ ID NO. 51.

In another preferred embodiment, the kit comprises a first container containing the set of PCR amplification primer pairs.

In another preferred embodiment, the kit comprises a second container containing the single base extension primer set.

In another preferred embodiment, the kit comprises a third container, wherein the third container contains a PCR premix liquid, and the PCR premix liquid mainly comprises hot start Taq enzyme, dNTPs and MgCl ₂ PCR buffer.

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

In another preferred embodiment, the kit comprises a fifth container containing SAP buffer.

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

In another preferred embodiment, the kit includes a seventh container containing ddNTPs.

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

In another preferred embodiment, the kit further comprises purified water.

In a second aspect of the invention, a method for detecting SNP gene mutation sites of diffuse toxic goiter susceptibility genes based on multiplex PCR time-of-flight mass spectrometry is provided, comprising the following steps:

(1) Taking the peripheral blood genome DNA of the sample to be detected as a template, and carrying out PCR amplification to obtain an amplification product;

(2) SAP processing is carried out on the amplified product in the step (1) by using 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 a desalting resin;

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

In another preferred embodiment, in the step (1), the SNP site selected from the group consisting of: rs2294025, rs7005834, rs2941522, rs907091, rs1453559, rs12150079, rs2872507, rs3087243, rs231775, rs229527, rs2284038, rs4969170, rs12101261, rs179243, rs40401, rs2069812, rs2227306.

In another preferred embodiment, in the step (1), the PCR amplification is performed using the amplification primer set during the PCR amplification.

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

In a third aspect of the invention, the use of a PCR amplification primer pair set is provided for preparing a detection kit for detecting SNP gene mutation sites of diffuse toxic goiter susceptibility genes;

the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 34.

In a fourth aspect of the invention, there is provided use of a single base extension primer set for preparing a detection kit for detecting a diffuse toxic goiter susceptibility gene SNP gene mutation site;

the single-base extension primer group comprises extension primers with sequences shown as SEQ ID NO.35 to SEQ ID NO. 51.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Detailed Description

The invention provides an early screening method and a kit for diffuse toxic goiter susceptibility genes, wherein 17 SNP loci of diffuse toxic goiter susceptibility genes of Chinese are screened out by comparing the difference of gene spectrums of Chinese population and European population, so that genetic markers related to diffuse toxic goiter can be screened and inspected widely (high-flux detection loci and high-flux detection samples) by using a nucleic acid mass spectrometer. Through multiple rounds of screening, the multiplex PCR amplification primer pair which can carry out high-efficiency multiplex amplification on the 17 SNP loci and is suitable for mass spectrum detection of Massarray nucleic acid is obtained, and the appropriate extension primer is obtained through screening, so that the high-accuracy and high-sensitivity detection on the 17 SNP loci is realized, the detection result is stable, and the detection positive rate is improved.

The determination method is based on a multiplex PCR technology and a Massary nucleic acid mass spectrometry technology to detect SNP gene mutation sites of diffuse toxic goiter susceptibility genes, and 17 sites can be detected simultaneously.

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

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

(1) The imbalance of the reaction system results in rapid amplification of certain advantageous primers and templates thereof in the previous rounds of reaction, resulting in large amounts of amplified products which are also good inhibitors of DNA polymerase. Therefore, with the large amount of amplified products, the polymerization ability of the polymerase is more and more strongly inhibited, and therefore, the primer and its template, which are at a disadvantage in the early stage, are more difficult to react, eventually resulting in an amount of amplified products that is too small to be detected.

(2) Primer specificity, if the primer binds more strongly to other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, resulting in a decrease in amplification efficiency.

(3) The optimal annealing temperatures are not uniform, and a plurality of pairs of primers are placed in a system for amplification, so that the optimal annealing temperature of each pair of primers is required to be close because the annealing temperatures for carrying out PCR reactions are the same.

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

Although several factors affecting amplification efficiency are mentioned above, more factors are not yet clear. To date, there is no effective method by which amplification efficiency can be predicted explicitly.

The multiplex PCR-time-of-flight mass spectrometry detection technology can detect with ultra-high throughput, but has high quality requirements on PCR amplification yield. The inventor finds that the existing amplification primer and extension primer capable of being detected by a multiplex fluorescence PCR method are directly applied to multiplex PCR-time-of-flight mass spectrometry detection, and have many defects, such as false negative of mass spectrometry detection caused by incapability of single base extension reaction, low sensitivity and poor repeatability, and are difficult to meet clinical application. Therefore, the inventor redesigns a plurality of pairs of amplification primers and extension primers for each detection site, performs multiple combination 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 results and are suitable for time-of-flight mass spectrometry detection through a large number of experimental screening.

The invention adopts a multiplex PCR method to amplify target sequences, designs a plurality of pairs of primers manually, performs optimization selection and verification on the primers, and finally determines a nucleic acid detection kit containing the amplification primers for detecting the total 17 sites of SNP gene mutation sites of diffuse toxic goiter susceptibility genes.

TABLE 1 amplification primers

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

The extension primers are shown in Table 2:

TABLE 2 extension primers

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

In addition to the amplification primer and the extension primer, the invention also provides a kit for detecting SNP gene mutation sites of diffuse toxic goiter susceptibility genes, and the specific content of each component in the detection kit is as follows:

TABLE 3 kit components

The invention also provides a method for detecting SNP gene mutation sites of diffuse toxic goiter susceptibility genes based on multiplex PCR time-of-flight mass spectrometry, which comprises the following steps:

(4) Purifying the extension product with a desalting resin;

Further, in the step (1), during the PCR amplification, SNP sites selected from the group consisting of: rs2294025, rs7005834, rs2941522, rs907091, rs1453559, rs12150079, rs2872507, rs3087243, rs231775, rs229527, rs2284038, rs4969170, rs12101261, rs179243, rs40401, rs2069812, rs2227306.

Further, in the step (1), the PCR amplification is performed using the amplification primer set during the 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: 95 ℃ for 3min;95 ℃, 15s,56 ℃, 15s,72 ℃, 1min,45 cycles; maintained at 72℃for 5min.

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

Further, the conditions of the extension reaction in the step (3) are as follows: 95 ℃ for 30s;95 ℃, 5s, (50 ℃, 5s,72 ℃, 5s,5 cycles), 35 cycles; maintained at 72℃for 5min.

The invention has the main advantages that:

the nucleic acid mass spectrometry early screening method of diffuse toxic goiter susceptibility genes provided by the invention considers the differences of diffuse toxic goiter gene spectrums of Chinese and European and American populations, detects susceptibility genes at the front edge, and comprises a plurality of SNP sites which are susceptible to diffuse toxic goiter in China, 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 the disadvantages of high price, long time consumption, complex operation and the like of the traditional single-base detection are changed. The sensitivity in early screening detection of diffuse toxic goiter susceptibility genes is higher, the flux is larger, the detection of single small sample polygene can be realized, and the maximization use of the small sample is satisfied.

The nucleic acid mass spectrometry method for detecting the diffuse toxic goiter susceptibility gene early sieve based on the Massary ARRAY nucleic acid mass spectrometry technology provided by the invention has the technical advantages of high accuracy and high sensitivity, has a stable 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 is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Example 1

Feasibility analysis of SNP locus screening of human diffuse toxic goiter susceptibility related genes

The present inventors have made screening and evaluation of diffuse toxic goiter occurrence related sites verified in large-scale pathology control clinical studies by searching NCBI domestic and foreign genome association analysis (genome-wide association study, GWAS), selected 17 single nucleotide polymorphism sites significantly related to the risk of diffuse toxic goiter occurrence in Chinese population, and independent of each other, without linkage disequilibrium, so that the site selection of the present invention has representativeness, independence and risk value accumulation, and can be used for evaluating the risk of diffuse toxic goiter occurrence in individuals.

The SNP loci screened were as follows:

rs2294025、rs7005834、rs2941522、rs907091、rs1453559、rs12150079、rs2872507、rs3087243、rs231775、rs229527、rs2284038、rs4969170、rs12101261、rs179243、rs40401、rs2069812、rs2227306。

example 2 System verification

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

Accuracy verification scheme: each site was detected in 20 cases, with an expected target of 95% compared to Sanger sequencing.

Specificity verification scheme: included in the accuracy, the target was expected to be 95%.

Sensitive verification scheme: the sensitivity investigation was performed with the positive samples of human genomic DNA as templates, and the DNA contents of the samples were calibrated to be 1 ng/. Mu.L, 5 ng/. Mu.L, 10 ng/. Mu.L, 50 ng/. Mu.L, and 100 ng/. Mu.L, respectively.

Precision validation protocol (including intra-batch, inter-batch, personnel alignment, not involving inter-instrument alignment) was expected to be 95%.

Precision in batch: each sample was repeated 3 times in the same batch and the precision in the batch was compared.

Precision between batches: the same operator tests the same sample in multiple batches and compares the precision between batches.

And (5) comparing the personnel: the same samples were tested by 2 operators and the differences in results between the comparison personnel were compared.

The specific test steps are as follows:

1. DNA extraction: blood DNA extraction kit (a commercially available kit for rapidly and efficiently extracting genomic DNA) autonomously developed according to southern core medicineThe procedure was followed to prepare human peripheral blood genomic DNA, 50. Mu.LddH ₂ Eluting with O;

2. PCR flow

(1) The sample was diluted to 20 ng/. Mu.L;

(2) A PCR reaction system (hereinafter, the single sample size, 40ng in total of sample DNA) was prepared as follows

TABLE 4 PCR reaction System

Reagent(s)	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 of	5.00	5.00

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

(4) The plates were put on a PCR instrument for the following thermal cycles:

95 ℃ for 3 minutes

45 cycles:

(95 ℃ C. For 15 seconds)

56 ℃ for 15 seconds

72 ℃ for 1 minute

72 ℃ for 5 minutes

Thermal insulation at 4 DEG C

2. SAP flow

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

(2) The SAP reaction system (hereinafter, single sample amount) was formulated as follows;

TABLE 5 SAP reaction System

Reagent(s)	Sample application per well (mu 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 of	2.00	4.00

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

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

(5) The plates were put on a PCR instrument for the following thermal cycles:

57 ℃ for 40 minutes

65 ℃ for 5 minutes

Thermal insulation at 4 DEG C

3. EXT (Single base extension) procedure

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

(2) EXT reaction systems (hereinafter, single sample amounts) were prepared as follows;

TABLE 6 EXT reaction System

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

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

(5) The plates were put on a PCR instrument for the following thermal cycles:

95℃30s

35 cycles:

(95℃、5s

5 cycles:

(50℃5s

72℃5s))

72℃5min

thermal insulation at 4 DEG C

4. Resin desalination

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

5. Dispensing sample application

Data were obtained using a MALDI-TOF (matrix assisted laser Desorption ionization-time of flight) mass spectrometer for each site cluster map (homogeneous cluster clarity).

Test results: the results of the accuracy verification are shown in table 7, taking 1 sample as an example.

TABLE 7 verification of accuracy (first generation sequencing vs Massary results)

SNP_ID	First generation sequencing results	Massary results
			rs2294025	GG	GG
rs7005834	CC	CC
			rs2941522	CT	CT
rs907091	CC	CC
			rs1453559	TT	TT
rs12150079	GG	GG
			rs2872507	GG	GG
rs3087243	GG	GG
			rs231775	AA	AA
rs229527	CC	CC
			rs2284038	AG	AG
rs4969170	AA	AA
			rs12101261	CC	CC
rs179243	TT	TT
			rs40401	CC	CC
rs2069812	AA	AA
			rs2227306	CC	CC

The sample rs229527 locus is exemplified in this example, and the results of the precision verification are shown in table 5.

Table 8 rs229527 site precision validation results

	Repeat 1	Repeat 2	Repeat 3
				Batch 1	CC	CC	CC
Batch 2	CC	CC	CC
				Batch 3	CC	CC	CC
Batch 4	CC	CC	CC
				Batch 5	CC	CC	CC

In the whole, all sites of the method are clustered clearly, basically have no gray area, and the false detection is possibly small. The detection accuracy (including sensitivity and specificity) and precision of each position point are verified in the application, and the detection accuracy and precision are shown in a table 9.

TABLE 9 results of accuracy, sensitivity and specificity verification

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In the above table, 100% accuracy indicates that all positive samples were correctly detected and consistent with Sanger sequencing results; 100% specificity indicates that no false positive results were present in the samples tested; the precision in the batch is 100% to show that the repeated detection results of the same batch of each sample can be kept consistent; the precision of 100% between batches indicates that the detection results of the same operator for checking the same sample in multiple batches can be kept consistent; personnel comparison of 100% shows that the detection results of the same sample can be kept consistent by 2 operators.

In conclusion, the nucleic acid mass spectrometry early screening method of the diffuse toxic goiter susceptibility gene provided by the invention considers the difference of diffuse toxic goiter gene spectra of Chinese and European and American populations, and the detected susceptibility gene is more advanced and comprises a plurality of SNP sites which are susceptible to diffuse toxic goiter, 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

For each site, the inventor designs a plurality to ten pairs of amplification primers and extension primers, and then verifies and optimizes the amplification primers and the extension primers, and finally establishes a multiplex PCR amplification primer and extension primer combination which can be used for detecting by a Massary ARRAY nucleic acid mass spectrometry technology.

This comparative example exemplifies amplification primers and extension primers with partial unsatisfactory effects, taking the rs2227306 locus as an example.

Control primer pair 1:

F-1：ACGTTGGATGCTGAAGGCAATTTCTATG(SEQ ID NO.:52)

R-1：ACGTTGGATGAAGGCATCATGTTTCTAG(SEQ ID NO.:53)

control primer pair 2:

F-2：ACGTTGGATGTTCTGAAGGGCAATTTCTATG(SEQ ID NO.:54)

R-2：ACGTTGGATGGGAAGGCATCATGTTTCT(SEQ ID NO.:55)

control primer pair 3:

F-3：ACGTTGGATGAGGCAATTTCTATGCTGG(SEQ ID NO.:56)

R-3：ACGTTGGATGAGCCCTTGACCTCAGTTAGT(SEQ ID NO.:57)

control extension primer 1:

Y-1：TCTAACTCTTTATATAGGAAGT(SEQ ID NO.:58)

control extension primer 2:

Y-2：TAACTCTTTATATAGGAAGT(SEQ ID NO.:59)

the primer pair of the invention: SEQ ID NOS.33 and 34

The extension primer of the invention: SEQ ID NO.51

Specific methods are the same as the above examples, in a single screening experiment, single PCR amplification is then performed using different extension primers for single base extension, and then mass spectrometry detection is performed on the extension products, and the single detection result shows that the detection of the control primer pair 1 and the control extension primer 1 in a single system can work normally, while positive results are obtained in a multiplex system.

In the multiplex system, the detection sensitivity of the extension primer combination shown in the control primer pairs 2 and 3 and SEQ ID NO.51 is 50 ng/. Mu.L and 50 ng/. Mu.L respectively; the combination of the control primer pair 2 and 3 and the control extension primer 2 has detection sensitivity of 50 ng/mu L and 10 ng/mu L respectively; the combination of the primers shown in SEQ ID NOS.33 and 34 and the control extension primer 2 gave a detection sensitivity of 10 ng/. Mu.L. The combination of the primer pair shown in SEQ ID No.33 and 34 and the extension primer shown in SEQ ID No.51 can achieve detection sensitivity of 5 ng/. Mu.L.

The results indicate that control primer pair 1 and control extension primer 1 are not effective in amplifying a target nucleic acid sequence in a detection system and therefore are not capable of working in a detection system; the control primer pairs 2 and 3 can work in a multiplex detection system, but have poorer sensitivity; control extension primer 2, although also operable during extension, is similarly less sensitive. The combination of the primer pair (SEQ ID NO.33 and 34) and the extension primer (SEQ ID NO. 51) can work normally in a multiplex detection system, and has high sensitivity reaching 5 ng/. Mu.L.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

acgttggatg agcctagacg atacac 26

<210> 4

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

acgttggatg agtcattcgt ccagaa 26

<210> 5

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

acgttggatg ttccgccacc acccaa 26

<210> 6

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

acgttggatg gaggcccgga ctaagcaa 28

<210> 7

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

acgttggatg gtcatttaga ttagggaga 29

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

acgttggatg agccagggaa acact 25

<210> 9

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

acgttggatg ctcaccttcc atgtcgc 27

<210> 10

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

acgttggatg cctgcccttt tcagtct 27

<210> 11

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

acgttggatg ttgagccagt tgatag 26

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

acgttggatg ggtttggtgg acaga 25

<210> 13

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

acgttggatg ctgcttacgt atcctg 26

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

acgttggatg acccctaaac ctatt 25

<210> 15

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

acgttggatg cattcagtat ctggtggag 29

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

acgttggatg gaaagcaggc ggtaa 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

acgttggatg acaccgctcc cataa 25

<210> 18

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

acgttggatg aaacacctcc tccatc 26

<210> 19

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

acgttggatg ggggtcctca gagtcac 27

<210> 20

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

acgttggatg aggcatcggc acatag 26

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

acgttggatg aggyycaggt ccatccc 27

<210> 22

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

acgttggatg tggcttcagg cacaaa 26

<210> 23

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

acgttggatg gacttctttc cattgtt 27

<210> 24

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

acgttggatg cctctatccc atctgt 26

<210> 25

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

acgttggatg atagtaggtg ccccaaac 28

<210> 26

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

acgttggatg ggcttcccaa agtgc 25

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

acgttggatg tatgctttgg gaaca 25

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

acgttggatg atggcaatct taggc 25

<210> 29

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

acgttggatg cccacgaagg accaga 26

<210> 30

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

acgttggatg caccagcaaa ggcaaa 26

<210> 31

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

acgttggatg atcctgggca cctt 24

<210> 32

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

acgttggatg accctggtca cagttca 27

<210> 33

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

acgttggatg taccctgatt atagacc 27

<210> 34

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

acgttggatg ctctgccagc tactt 25

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

acctttcctc acccagaaat 20

<210> 36

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

cgatgcgtat gtagggtt 18

<210> 37

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

gccgcagcaa cgagc 15

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

tgtttcatat agcacatctc 20

<210> 39

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

tgccgctgtg gctac 15

<210> 40

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

accaaattgt aggacgtt 18

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

tactgcttat gtatcctgcc 20

<210> 42

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

tcaccactat ttgggatata ac 22

<210> 43

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

ctcagctgaa cctggct 17

<210> 44

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

cccagggcgg ctgtc 15

<210> 45

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

gcccagaagc tctctcc 17

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

cattgttttt agagaccaca 20

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

gtcctttgca tctttttcta 20

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

agaaaggaaa aattgtaact 20

<210> 49

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

ccatgaccca gacaacg 17

<210> 50

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

tgtccagact cctggatct 19

<210> 51

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 51

ctaactcttt atataggaag t 21

<210> 52

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 52

acgttggatg ctgaaggcaa tttctatg 28

<210> 53

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

acgttggatg aaggcatcat gtttctag 28

<210> 54

<211> 31

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 54

acgttggatg ttctgaaggg caatttctat g 31

<210> 55

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

acgttggatg ggaaggcatc atgtttct 28

<210> 56

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 56

acgttggatg aggcaatttc tatgctgg 28

<210> 57

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

acgttggatg agcccttgac ctcagttagt 30

<210> 58

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 58

tctaactctt tatataggaa gt 22

<210> 59

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

taactcttta tataggaagt 20

Claims

1. An early screening kit for diffuse toxic goiter susceptibility genes, characterized in that the kit comprises a PCR amplification primer pair group and a single base extension primer group, wherein the PCR amplification primer pair group comprises a primer pair for specifically amplifying the following SNP sites: rs2294025, rs7005834, rs2941522, rs907091, rs1453559, rs12150079, rs2872507, rs3087243, rs231775, rs229527, rs2284038, rs4969170, rs12101261, rs179243, rs40401, rs2069812 and rs2227306;

wherein, in the PCR amplification primer pair group:

the primer pair for specifically amplifying rs2294025 is shown as SEQ ID NO.1 and SEQ ID NO. 2;

the primer pair for specifically amplifying rs7005834 is shown as SEQ ID NO.3 and SEQ ID NO. 4;

the primer pair for specifically amplifying rs2941522 is shown as SEQ ID NO.5 and SEQ ID NO. 6;

the primer pair for specifically amplifying rs907091 is shown as SEQ ID NO.7 and SEQ ID NO. 8;

the primer pair for specifically amplifying rs1453559 is shown as SEQ ID NO.9 and SEQ ID NO. 10;

the primer pair for specifically amplifying rs12150079 is shown as SEQ ID NO.11 and SEQ ID NO. 12;

the primer pair for specifically amplifying rs2872507 is shown as SEQ ID NO.13 and SEQ ID NO. 14;

the primer pair for specifically amplifying rs3087243 is shown as SEQ ID NO.15 and SEQ ID NO. 16;

the primer pair for specifically amplifying rs231775 is shown as SEQ ID NO.17 and SEQ ID NO. 18;

the primer pair for specifically amplifying rs229527 is shown as SEQ ID NO.19 and SEQ ID NO. 20;

the primer pair for specifically amplifying rs2284038 is shown as SEQ ID NO.21 and SEQ ID NO. 22;

the primer pair for specifically amplifying rs4969170 is shown as SEQ ID NO.23 and SEQ ID NO. 24;

the primer pair for specifically amplifying rs12101261 is shown as SEQ ID NO.25 and SEQ ID NO. 26;

the primer pair for specifically amplifying rs179243 is shown as SEQ ID NO.27 and SEQ ID NO. 28;

the primer pair for specifically amplifying rs40401 is shown as SEQ ID NO.29 and SEQ ID NO. 30;

the primer pair for specifically amplifying rs2069812 is shown as SEQ ID NO.31 and SEQ ID NO. 32;

the primer pair for specifically amplifying rs2227306 is shown as SEQ ID NO.33 and SEQ ID NO. 34;

the single base extension primer set comprises:

the extended primer for rs2294025 is shown in SEQ ID NO. 35;

the extended primer for rs7005834 is shown in SEQ ID NO. 36;

the extended primer for rs2941522 is shown in SEQ ID NO. 37;

the extended primer for rs907091 is shown in SEQ ID NO. 38;

the extended primer for rs1453559 is shown in SEQ ID NO. 39;

the extended primer for rs12150079 is shown in SEQ ID NO. 40;

the extended primer for rs2872507 is shown in SEQ ID NO. 41;

the extended primer for rs3087243 is shown in SEQ ID NO. 42;

the extended primer for rs231775 is shown in SEQ ID NO. 43;

the extended primer for rs229527 is shown in SEQ ID NO. 44;

the extended primer for rs2284038 is shown in SEQ ID NO. 45;

the extended primer for rs4969170 is shown as SEQ ID NO. 46;

the extended primer for rs12101261 is shown in SEQ ID NO. 47;

the extension primer for rs179243 is shown in SEQ ID NO. 48;

the extension primer for rs40401 is shown in SEQ ID NO. 49;

the extension primer for rs2069812 is shown as SEQ ID NO. 50;

the extended primer for rs2227306 is shown as SEQ ID NO. 51.

2. The kit of claim 1, wherein the kit comprises a first container containing the set of PCR amplification primer pairs therein.

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

4. A reagent according to claim 3The kit is characterized by further comprising a third container, wherein the third container contains PCR premix liquid, and the PCR premix liquid comprises hot start Taq enzyme, dNTPs and MgCl ₂ And PCR buffer.

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

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

7. The kit of claim 6, wherein the kit comprises a sixth container comprising an elongase therein.

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

9. The kit of claim 8, wherein the kit comprises an eighth container containing an extension reaction buffer.

10. The kit of claim 9, further comprising purified water.