CN116536413A - SNP locus combination, product and method for detecting genetic susceptibility of gestational diabetes - Google Patents

SNP locus combination, product and method for detecting genetic susceptibility of gestational diabetes Download PDF

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CN116536413A
CN116536413A CN202310450993.1A CN202310450993A CN116536413A CN 116536413 A CN116536413 A CN 116536413A CN 202310450993 A CN202310450993 A CN 202310450993A CN 116536413 A CN116536413 A CN 116536413A
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snp
seq
snp locus
gestational diabetes
gene
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郭润民
曾巧莉
卫月
杨太丽
邵滔滔
张恒丽
刘佳
刘鑫
刘嘉欣
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Shunde Women's And Children's Hospital Of Guangdong Medical University
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Abstract

The invention relates to the technical field of molecular diagnosis, in particular to SNP locus combination, product and method for detecting genetic susceptibility of gestational diabetes, wherein the SNP locus combination specifically comprises SNP locus 1: the position is rs1552224 on ARAP1 gene; SNP site 2: rs937301 located on GIP gene; SNP site 3: rs3848460 located on GIP gene; SNP site 4: rs11705701 located on IGF2BP2 gene. Meanwhile, the product provided by the invention is used for specifically detecting the SNP locus combination, and the method can effectively fill the gap of the prior art in the aspect of gestational diabetes genetic susceptibility detection, and improve the accuracy of gestational diabetes genetic susceptibility detection.

Description

SNP locus combination, product and method for detecting genetic susceptibility of gestational diabetes
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to SNP locus combination, a product and a method for detecting genetic susceptibility of gestational diabetes.
Background
Gestational diabetes mellitus (Gestational diabetes mellitus, GDM) is a multifactorial etiologic disease caused by insulin resistance and impaired insulin secretion. There is increasing evidence that genetic factors contribute to GDM susceptibility. Furthermore, genetic variations associated with insulin resistance and β -cell dysfunction are believed to play a key role in the development of GDM. Studies have shown an increased risk of developing type two diabetes (T2 DM) in women with a history of GDM, and a possible increase in the risk of developing GDM in women with a family history of diabetes. There is growing evidence that genetic variation of genes in the metabolic pathway of diabetes may increase the risk of susceptibility to diabetes, and that such polymorphisms may affect the normal expression of genes, further increasing the risk of GDM. Therefore, in order to prevent the occurrence of GDM, it is necessary to detect Single Nucleotide Polymorphism (SNPs) variation.
However, in the current similar technology, most of the technologies are aimed at single SNP loci, and few are aimed at SNP loci on two genes, so that the prediction result is limited and the practical application is not strong. The method can not provide a more powerful and wider technical means for screening high-risk groups of gestational diabetes mellitus, so that the GDM is difficult to realize early detection and early interference.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide SNP locus combinations, products and methods for detecting genetic susceptibility of gestational diabetes, so as to solve the defects in the prior art.
In order to achieve the technical effects, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a SNP site combination for detecting gestational diabetes genetic susceptibility, the SNP site combination comprising 4 SNP sites, specifically SNP site 1, SNP site 2, SNP site 3 and SNP site 4, wherein:
SNP locus 1 is rs1552224 on ARAP1 gene;
SNP locus 2 is rs937301 located on the GIP gene;
SNP locus 3 is rs3848460 located on the GIP gene;
SNP site 4 is rs11705701 located on the IGF2BP2 gene.
In a second aspect, the invention also provides an application of the SNP locus combination for detecting genetic susceptibility of gestational diabetes provided in the first aspect in preparing a product for predicting gestational diabetes mellitus risk and/or in preparing a product for screening or assisting in screening gestational diabetes mellitus.
Further, the application comprises the steps of:
obtaining a biological sample of a subject, extracting sample DNA of the biological sample;
detecting the base sequences of the sample DNA at the SNP locus 1, the SNP locus 2, the SNP locus 3 and the SNP locus 4, and respectively determining the genotypes of the subjects at the SNP locus 1, the SNP locus 2, the SNP locus 3 and the SNP locus 4 according to the base sequences;
combining the detection results of genotypes of the 4 SNP loci to distinguish whether the subjects belong to genetic high-risk groups or genetic low-risk groups;
preferably, the subject is a asian population, further preferably, the asian population is a chinese.
Preferably, the biological sample is derived from any one of human blood, feces, urine, oral epithelial cells, exfoliated cells, oral swab, or throat swab.
In a third aspect, the present invention also provides a specific amplification primer pair for detecting the SNP site combination as set forth in claim 1, or a specific fluorescent probe pair for detecting the SNP site combination as set forth in claim 1.
In a fourth aspect, the present invention also provides the use of a specific amplification primer pair or a specific fluorescent probe pair as provided in the third aspect above, in particular in the manufacture of a product for predicting the risk of gestational diabetes and/or in the manufacture of a product for screening or aiding in the screening of gestational diabetes.
In a fifth aspect, the present invention still further provides a product for detecting genetic susceptibility to gestational diabetes, the product comprising a pair of specific amplification primers for detecting 4 SNP sites in a SNP site combination, respectively, and/or a pair of specific fluorescent probes;
wherein, the 4 SNP loci are respectively:
SNP locus 1 is rs1552224 on ARAP1 gene;
SNP locus 2 is rs937301 located on the GIP gene;
SNP locus 3 is rs3848460 located on the GIP gene;
SNP locus 4 is rs11705701 located on IGF2BP2 gene;
preferably, the product is a kit.
Preferably, the four sets of specific amplification primer pairs comprise:
a specific primer pair 1 comprising a nucleotide sequence as set forth in SEQ ID NO:1 and the sequence of SEQ ID NO:1, wherein the specific primer pair 1 is used for specifically detecting the genotype of the SNP locus 1;
a specific primer pair 2 comprising a nucleotide sequence as set forth in SEQ ID NO:3 and the upstream primer shown in SEQ ID NO:4, the specific primer pair 2 is used for specifically detecting the genotype of the SNP locus 2;
a specific primer pair 3 comprising a nucleotide sequence as set forth in SEQ ID NO:5 and the sequence of SEQ ID NO:6, the specific primer pair 3 is used for specifically detecting the genotype of the SNP locus 3;
a specific primer pair 4 comprising a nucleotide sequence as set forth in SEQ ID NO:7 and the upstream primer shown in SEQ ID NO:8, and the specific primer pair 4 is used for specifically detecting the genotype of the SNP site 4.
Preferably, the four sets of specific fluorescent probe pairs comprise:
a specific fluorescent probe pair 01 comprising a nucleotide sequence as set forth in SEQ ID NO:9, and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:10, a fluorescent gene probe with FAM, wherein the specific fluorescent probe pair 01 is used for specifically detecting the genotype of SNP locus 1;
a specific fluorescent probe pair 02 comprising a nucleotide sequence as set forth in SEQ ID NO:11 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:12, a fluorescent gene probe with FAM, wherein the specific fluorescent probe pair 01 is used for specifically detecting the genotype of the SNP locus 2;
a specific fluorescent probe pair 03 comprising a nucleotide sequence as set forth in SEQ ID NO:13 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:14, wherein the specific fluorescent probe pair 01 is used for specifically detecting the genotype of the SNP locus 3;
a specific fluorescent probe pair 04 comprising a nucleotide sequence as set forth in SEQ ID NO:15 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:16, and the specific fluorescent probe pair 01 is used for specifically detecting the genotype of the SNP locus 4.
Preferably, the product further comprises one or more of a ribozyme-free water, a buffer, a dNTP mix, a DNA polymerase.
In a sixth aspect, the present invention still further provides a method for detecting genetic susceptibility to gestational diabetes, the method comprising the steps of:
obtaining a biological sample of a subject, extracting sample DNA of the biological sample;
detecting genotypes of 4 SNP loci in the SNP locus combination respectively by using the product for detecting genetic susceptibility of gestational diabetes according to any one of claims 6 to 9;
and (3) combining the detection results of genotypes of the 4 SNP loci to distinguish whether the subjects belong to genetic high-risk groups or genetic low-risk groups.
Preferably, the subject is a asian population, further preferably, the asian population is a chinese.
Compared with the prior art, the invention has the beneficial effects that:
first, the present invention proposes the detection of SNP locus combinations for detecting genetic susceptibility of gestational diabetes for the first time. The SNP locus combination specifically comprises SNP locus 1: the position is rs1552224 on ARAP1 gene; SNP site 2: rs937301 located on GIP gene; SNP site 3: rs3848460 located on GIP gene; SNP site 4: rs11705701 located on IGF2BP2 gene; meanwhile, the invention also provides a product for detecting genetic susceptibility of gestational diabetes, which is used for specifically detecting the SNP locus combination. In addition, the SNP locus combination for detecting genetic susceptibility of gestational diabetes is based on the synergistic effect among all SNP loci in the SNP locus combination, and can effectively and accurately screen genetic high risk groups of gestational diabetes by detecting genotypes of the SNP locus combination, thereby providing a more powerful and wider technical means for early detection of gestational diabetes and solving the defects in the prior art.
Detailed Description
Embodiments of the present invention will be described in detail below. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents or apparatus used, unless specifically stated otherwise, are those commercially available and the specific conditions in the examples are as per conventional conditions or as recommended by the manufacturer.
Example 1
The results of genotyping 500 GDM patients and 502 healthy pregnant women while calculating the OR values of the allele model, dominant model, recessive model, homozygous model, heterozygous model and super dominant model at SNP site 1, SNP site 2, SNP site 3 and SNP site 4 are shown in table 1:
TABLE 1 correlation results of the risk of onset of GDM with each SNP site of the SNP combination provided by the invention
From the results of Table 1, it was found that the wild-type alleles at SNP site 1, SNP site 2 and SNP site 3 significantly increased the risk of GDM onset, and that mutant alleles have a protective effect on GDM. SNP site 4 mutant alleles significantly increase the risk of GDM onset, and the synergistic effect of the 4 SNP sites finds that haplotype C-A-A-G can significantly reduce the risk of GDM onset, and the statistical results are shown in Table 2:
correlation of haplotypes of SNPs in Table 2 with risk of gestational diabetes onset
Example 2
The embodiment provides a kit for detecting genetic susceptibility of gestational diabetes, which comprises four groups of specific amplification primer pairs and four groups of specific fluorescent probe pairs, wherein the four groups of specific amplification primer pairs comprise:
a specific primer pair 1 comprising a nucleotide sequence as set forth in SEQ ID NO:3 and the upstream primer shown in SEQ ID NO: 4;
a specific primer pair 3 comprising a nucleotide sequence as set forth in SEQ ID NO:5 and the sequence of SEQ ID NO:6, a downstream primer shown in FIG. 6;
a specific primer pair 4 comprising a nucleotide sequence as set forth in SEQ ID NO:7 and the upstream primer shown in SEQ ID NO: 8.
The four sets of specific fluorescent probe pairs include:
a specific fluorescent probe pair 01 comprising a nucleotide sequence as set forth in SEQ ID NO:9, and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:10 with FAM fluorescent gene probe;
a specific fluorescent probe pair 02 comprising a nucleotide sequence as set forth in SEQ ID NO:11 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:12 with FAM fluorescent gene probe;
a specific fluorescent probe pair 03 comprising a nucleotide sequence as set forth in SEQ ID NO:13 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:14 with FAM fluorescent gene probe;
a specific fluorescent probe pair 04 comprising a nucleotide sequence as set forth in SEQ ID NO:15 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:16 with FAM fluorescent gene probe.
Meanwhile, the kit provided by the invention comprises non-ribozyme water, buffer solution, dNTP mixed solution and DNA polymerase.
In addition, based on the above kit, the present embodiment further provides a method for detecting genetic susceptibility of gestational diabetes using the kit, the method comprising the steps of:
s1: obtaining a biological sample of a subject, and extracting sample DNA of the biological sample, wherein the specific operations are as follows:
genomic DNA from whole blood (and also oral epithelial cells and other biological sample DNA) was extracted with silica-hydroxymagnetic beads and manipulated according to known methods.
S2: fluorescent quantitative PCR reactions and result reads to determine subject genotypes
The genotypes of 4 SNP loci in the SNP locus combination are respectively detected by using the kit through fluorescent quantitative PCR reaction, and the method comprises the steps of adopting a specific primer pair 1 and a specific fluorescent probe pair 01 to specifically detect the genotype of the SNP locus 1; the genotype of SNP locus 2 is detected specifically by adopting a specific primer pair 2 and a specific fluorescent probe pair 02; the genotype of SNP locus 3 is detected by adopting a specific primer pair 3 and a specific fluorescent probe pair 03; the genotype of SNP locus 4 is detected specifically by adopting a specific primer pair 4 and a specific fluorescent probe pair 04; the specific operation method is as follows:
the SNP site 1, SNP site 2, SNP site 3 and SNP site 4 were subjected to fluorescent quantitative PCR reactions respectively, each of which was carried out in a total volume of 20. Mu.L, 10. Mu.L of Probe qPCR Mix (2X), 1. Mu.L of DNA template (50 ng/. Mu.L), 1. Mu.L of upstream primer, 1. Mu.L of downstream primer (10. Mu.M) and 1. Mu.L of fluorescent Probe (10. Mu.M) were each carried out, and the above-mentioned systems were placed on an ABI Quantum studio type 5 PCR amplification apparatus to carry out the reactions under the following reaction conditions:
hot start at 95 ℃ for 10 min; 95 ℃,10 s,60 ℃,45 s,40 cycles; fluorescence signals were collected at the end of 60℃and read on an ABI Quantum studio type 5 PCR instrument after the end of the reaction.
The person skilled in the art of fluorescent quantitative PCR technology determines the genotype of a subject by identifying the amount of fluorescence of the final sample displayed on a fluorescent quantitative PCR instrument and determining the genotype of the SNP locus detected according to the intensity of the fluorescent signals of the probes VIC and FAM of different sequences.
Example 3
The embodiment provides a method for detecting gestational diabetes genetic susceptibility of a pregnant woman, comprising the steps of:
s31: obtaining a biological sample from a subject, which sample may be any sample comprising genomic DNA, such as a whole blood sample or an oral epithelial cell sample, preferably a whole blood sample;
genomic DNA from whole blood was extracted with silica-hydroxymagnetic beads and subjected to a known method.
S32: genotyping assays
By using the detection kit provided by the invention (the composition of the kit and the use method refer to example 2), the SNP locus 1, the SNP locus 2, the SNP locus 3 and the SNP locus 4 of the genomic DNA of a detected subject are respectively subjected to fluorescent quantitative PCR detection, and the genotypes of the 4 SNP loci in the SNP locus combination are determined.
S33: analysis of genetic susceptibility to gestational diabetes
By analyzing SNP genotypes of the detected subjects, analysis report forms of genetic susceptibility of gestational diabetes are provided, the genetic susceptibility of the detected subjects is specified in the report, and doctors specify and read the genetic susceptibility analysis report forms to the detected subjects.
However, it should be noted that the above-described genetic susceptibility detection result is not equivalent to the diagnosis result of the disease.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.

Claims (10)

1. A SNP locus combination for detecting genetic susceptibility of gestational diabetes, characterized by comprising 4 SNP loci, wherein,
SNP locus 1 is rs1552224 on ARAP1 gene;
SNP locus 2 is rs937301 located on the GIP gene;
SNP locus 3 is rs3848460 located on the GIP gene;
SNP site 4 is rs11705701 located on the IGF2BP2 gene.
2. Use of a combination of SNP sites for detecting gestational diabetes genetic susceptibility as defined in claim 1 for the manufacture of a product for predicting gestational diabetes mellitus risk and/or for the manufacture of a product for screening or assisting in screening gestational diabetes.
3. The use according to claim 2, comprising the steps of:
obtaining a biological sample of a subject, extracting sample DNA of the biological sample;
detecting the base sequences of the sample DNA at the SNP locus 1, the SNP locus 2, the SNP locus 3 and the SNP locus 4, and respectively determining the genotypes of the subjects at the SNP locus 1, the SNP locus 2, the SNP locus 3 and the SNP locus 4 according to the base sequences;
combining the detection results of genotypes of the 4 SNP loci to distinguish whether the subjects belong to genetic high-risk groups or genetic low-risk groups;
preferably, the subject is a asian population, further preferably, the asian population is a chinese.
4. A use according to claim 3, wherein: the biological sample is derived from any one of human blood, feces, urine, oral epithelial cells, exfoliated cells, oral swab, or throat swab.
5. Use of a specific amplification primer pair for detecting a SNP site combination as defined in claim 1 and/or a specific fluorescent probe pair for detecting a SNP site combination as defined in claim 1 for the preparation of a product for predicting the risk of gestational diabetes mellitus and/or for the preparation of a product for screening or assisting in screening gestational diabetes mellitus.
6. A product for detecting genetic susceptibility to gestational diabetes, comprising a pair of specific amplification primers for detecting 4 SNP sites in a SNP site combination, respectively, and/or a pair of specific fluorescent probes;
wherein, the 4 SNP loci are respectively:
SNP locus 1 is rs1552224 on ARAP1 gene;
SNP locus 2 is rs937301 located on the GIP gene;
SNP locus 3 is rs3848460 located on the GIP gene;
SNP locus 4 is rs11705701 located on IGF2BP2 gene;
preferably, the product is a kit.
7. A product for detecting genetic susceptibility to gestational diabetes as claimed in claim 6, wherein the four sets of specific amplification primer pairs comprise:
a specific primer pair 1 comprising a nucleotide sequence as set forth in SEQ ID NO:1 and the sequence of SEQ ID NO:1, a downstream primer shown in FIG. 1;
a specific primer pair 2 comprising a nucleotide sequence as set forth in SEQ ID NO:3 and the upstream primer shown in SEQ ID NO: 4;
a specific primer pair 3 comprising a nucleotide sequence as set forth in SEQ ID NO:5 and the sequence of SEQ ID NO:6, a downstream primer shown in FIG. 6;
a specific primer pair 4 comprising a nucleotide sequence as set forth in SEQ ID NO:7 and the upstream primer shown in SEQ ID NO: 8.
8. A product for detecting genetic susceptibility to gestational diabetes as claimed in claim 6, wherein the four sets of specific fluorescent probe pairs comprise:
a specific fluorescent probe pair 01 comprising a nucleotide sequence as set forth in SEQ ID NO:9, and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:10 with FAM fluorescent gene probe;
a specific fluorescent probe pair 02 comprising a nucleotide sequence as set forth in SEQ ID NO:11 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:12 with FAM fluorescent gene probe;
a specific fluorescent probe pair 03 comprising a nucleotide sequence as set forth in SEQ ID NO:13 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:14 with FAM fluorescent gene probe;
a specific fluorescent probe pair 04 comprising a nucleotide sequence as set forth in SEQ ID NO:15 and the nucleotide sequence of the fluorescent gene probe with VIC is shown as SEQ ID NO:16 with FAM fluorescent gene probe.
9. A product for detecting genetic susceptibility to gestational diabetes as claimed in claim 6, wherein: the product also comprises one or more of non-ribozyme water, buffer solution, dNTP mixed solution and DNA polymerase.
10. A method for detecting genetic susceptibility to gestational diabetes, comprising the steps of:
obtaining a biological sample of a subject, extracting sample DNA of the biological sample;
detecting genotypes of 4 SNP loci in the SNP locus combination respectively by using the product for detecting genetic susceptibility of gestational diabetes according to any one of claims 6 to 9;
and (3) combining the detection results of genotypes of the 4 SNP loci to distinguish whether the subjects belong to genetic high-risk groups or genetic low-risk groups.
CN202310450993.1A 2023-04-25 2023-04-25 SNP locus combination, product and method for detecting genetic susceptibility of gestational diabetes Pending CN116536413A (en)

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