阅读说明：本技术 用于检测尼古丁依赖相关的snp位点的引物组、应用、产品及方法 (Primer group, application, product and method for detecting nicotine dependence related SNP site ) 是由 李萌 夭建华 朱洲海 管莹 陆舍铭 徐玉琼 彭琪媛 唐萍 缪明明 陈章玉 李雪梅 于 2020-06-28 设计创作，主要内容包括：本发明涉及用于检测尼古丁依赖相关的SNP位点的引物组、应用、产品及方法,属于生物技术领域。所述的SNP位点位于人基因组第9号染色体的8561542至9916353区间段,位于PTPRD基因的内含子区间。本发明提供了用于检测这些SNP位点的引物对,以及一种鉴定它们等位基因型的简便方法。同时提供了检测上述SNP位点的试剂在制备用于检测尼古丁依赖易感性的检测制剂或检测装置中的应用。本发明对检测中国人群个体的尼古丁依赖易感性具有重要参考价值,在将来开发预防尼古丁依赖的药物过程中也有广阔的应用前景。(The invention relates to a primer group, application, a product and a method for detecting nicotine dependence related SNP sites, and belongs to the technical field of biology. The SNP locus is located in the section from 8561542 to 9916353 of chromosome 9 of the human genome and in the intron section of the PTPRD gene. The present invention provides primer pairs for detecting these SNP sites, and a simple method for identifying their allelic forms. Simultaneously provides the application of the reagent for detecting the SNP locus in the preparation of a detection preparation or a detection device for detecting the nicotine dependence susceptibility. The invention has important reference value for detecting the susceptibility of individual nicotine dependence of Chinese population, and has wide application prospect in the process of developing the medicament for preventing nicotine dependence in the future.)

1. A primer group for detecting nicotine dependence related SNP sites, which is characterized in that the SNP sites corresponding to the primer group are rs71507664, rs35841018, rs115729775, rs77835962, rs35831393, rs71497118, rs71492010, rs76717037, rs12347862, rs1323791, rs10816208, rs10816209, rs10816210, rs10978037, rs10978038, rs10978039, rs1408117, rs1408118, rs10816211, rs10816212, rs10816213, rs10978042, rs10978043, rs12380595, rs72692730, rs10978078, rs 72276931, rs72692733, rs72692735 and rs 72276936;

the primer group comprises 30 pairs of primer pairs, and the primer pairs have nucleotide sequences shown in SEQ ID NO. 1-60.

2. Use of the primer set for detecting a nicotine dependence-associated SNP site of claim 1 in the preparation of a product for detecting nicotine dependence susceptibility.

3. A product for detecting a susceptibility to nicotine dependence, the product comprising the primer set of claim 1.

4. A nicotine-dependent product according to claim 3, further comprising a reagent for detecting the SNP site of claim 1.

5. Use of a reagent for detecting a plurality of single nucleotide polymorphism sites for the manufacture of a detection preparation or a detection device for detecting susceptibility to nicotine dependence, characterized in that: the plurality of single nucleotide polymorphic sites comprises the following 30 single nucleotide polymorphic sites:

rs71507664, rs35841018, rs115729775, rs77835962, rs35831393, rs71497118, rs71492010, rs76717037, rs12347862, rs1323791, rs10816208, rs10816209, rs10816210, rs10978037, rs10978038, rs10978039, rs1408117, rs1408118, rs10816211, rs10816212, rs10816213, rs10978042, rs10978043, rs12380595, rs72692730, rs10978078, rs72692731, rs72692733, rs72692735 and rs 72276936.

6. Use of the reagent for detecting a plurality of single nucleotide polymorphism sites according to claim 5, in the preparation of a detection preparation or a detection device for detecting nicotine dependency susceptibility, characterized in that:

the rs71507664 allele type is A, rs35841018 allele type, the T, rs115729775 allele type is A, rs77835962 allele type, the G, rs35831393 allele type is C, rs71497118 allele type, the C, rs71492010 allele type is A, rs76717037 allele type, the G, rs12347862 allele type is C, rs1323791 allele type, the G, rs 16208 allele type is C, rs10816209 allele type, the G, rs 16210 allele type is C, rs10978037 allele type, the A, rs10978038 allele type is A, rs10978039 allele type, the 361401408117 allele type is 361401401401401401408118 allele type is C, rs 16211 allele type, the C, rs 7272727272727272699 allele type is 3616213 allele type, the C, rs allele type is 3678042 allele type, the 362769595 2769595 279 allele type, the 362769595 279 allele type, the 3627729 allele type, the 36277269595 7272 allele type, the 36277272729 allele type, the 362769595 729 allele type, the 362769595 729 type, the allele type, the, The rs72692736 allele type is T.

7. Use of the reagent for detecting a plurality of single nucleotide polymorphism sites according to claim 5, in the preparation of a detection preparation or a detection device for detecting nicotine dependency susceptibility, characterized in that: the sample to be tested is derived from blood, urine, saliva, gastric juice, hair or biopsy of the subject to be tested.

8. A method for detecting a nicotine dependence-associated SNP site genotype, comprising the steps of:

(1) using human whole genome DNA to be detected as a template, and respectively adopting 30 pairs of primers shown by SEQ ID NO.1-60 to carry out PCR amplification to obtain amplified fragments;

(2) and carrying out agarose gel electrophoresis on the amplified fragment to separate a target band, cutting the gel to recover a product, and then judging the genotype of the nicotine-dependent SNP locus by using a sanger sequencing method.

9. The method for detecting the genotype of a nicotine-dependent SNP site according to claim 8, wherein the PCR amplification system is:

1 μ L of Taq DNA polymerase, 10 pmol/. mu.L of forward primer 0.6 μ L, 10 pmol/. mu.L of reverse primer 0.6 μ L, 2 μ L of template DNA, 2 μ L of 10 XBuffer, 2.5mM of dNTP 1.6mL, ddH₂O12.2. mu.L, 20. mu.L in total.

10. The method for detecting the genotype of a nicotine-dependent SNP site according to claim 8, wherein the PCR amplification procedure is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ or annealing at the annealing temperature in Table 1 for 45s, and extension at 72 ℃ for 45s for 45 cycles; final extension at 72 deg.C for 5min, and storage at 4 deg.C.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a primer set, application, a product and a method for detecting nicotine dependence related SNP sites.

Background

Smoking behavior is not only closely related to the living environment, but also affected by genetic factors. Genetically, smoking behavior may be associated with polymorphisms in various genes of the neurotransmitter system, metabolic enzymes, and the like. Several studies have identified a number of genomic regions associated with smoking behavior, which are located on chromosomes 3-7, 9-11, 17, 20, and 22, respectively. Nicotine is a chemical present in tobacco, and the development of nicotine dependence is an important factor affecting the continuous smoking of smokers. Studies have shown that nicotine dependence is affected by genes such as nicotinic acetylcholine receptors, dopamine transporter proteins, and serotonin receptors.

Protein tyrosine phosphatase is a signaling molecule that regulates cell growth, differentiation, the mitotic cycle. The receptor type protein tyrosine phosphatase D (PTPRD) gene has important influence on the generation of nicotine dependence, for example, SNPs rs 1975197, rs4626664 and rs2381970 on the PTPRD gene can influence the degree of nicotine dependence by changing the expression of the PTPRD gene. However, there is little evidence for the site of a nicotine-dependent SNP in the PTPRD gene, and only a single SNP has sufficient evidence to support the conclusion of a nicotine-dependent event. In addition, no report has been made on how to simply and efficiently obtain a sequence containing a nicotine-dependent SNP site on the PTPRD gene by PCR.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a primer group, application, a product and a method for detecting nicotine dependence related SNP sites.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a primer group for detecting nicotine dependence related SNP sites, wherein the SNP sites corresponding to the primer group are rs71507664, rs35841018, rs115729775, rs77835962, rs35831393, rs71497118, rs71492010, rs76717037, rs12347862, rs1323791, rs10816208, rs10816209, rs10816210, rs10978037, rs10978038, rs10978039, rs1408117, rs1408118, rs10816211, rs10816212, rs10816213, rs10978042, rs10978043, rs 80123595, rs 109272730, rs10978078, rs 722731, rs 72276933, rs 72276935 and rs 72692736;

the primer group comprises 30 pairs of primer pairs, and the primer pairs have nucleotide sequences shown in SEQ ID NO. 1-60.

The invention also provides application of the primer group for detecting the nicotine dependence related SNP locus in preparing products for detecting nicotine dependence susceptibility.

The invention provides a product for detecting nicotine dependence susceptibility, which comprises the primer group. The product can be a reagent, a kit, a detection device and the like.

Further, it is preferable that the product further comprises a reagent for detecting the SNP site.

The present invention further provides use of an agent for detecting a plurality of single nucleotide polymorphic sites in the manufacture of a detection preparation or a detection apparatus for detecting susceptibility to nicotine dependence, the plurality of single nucleotide polymorphic sites comprising the following 30 single nucleotide polymorphic sites:

rs71507664, rs35841018, rs115729775, rs77835962, rs35831393, rs71497118, rs71492010, rs76717037, rs12347862, rs1323791, rs10816208, rs10816209, rs10816210, rs10978037, rs10978038, rs10978039, rs1408117, rs1408118, rs10816211, rs10816212, rs10816213, rs10978042, rs10978043, rs12380595, rs72692730, rs10978078, rs72692731, rs72692733, rs72692735 and rs 72276936.

Further, it is preferred that the rs71507664 allele type is A, rs35841018 allele type, T, rs115729775 allele type, A, rs77835962 allele type, G, rs35831393 allele type, C, rs71497118 allele type, C, rs71492010 allele type, A, rs76717037 allele type, G, rs 47862 allele type, C, rs 13213291 allele type, G, rs 16208 allele type, C, rs10816209 allele type, G, rs10816210 allele type, C, rs10978037 allele type, A, rs10978038 allele type, A, rs10978039 allele type, 861408117 allele type, C, rs1408118 allele type, T, rs 1627272727211 allele type, 3616212 allele type, G, rs allele type, 362769595 276972 type, 36277872 allele type, 3627782 allele type, 3627783 allele type, 36277272 allele type, 3627729 allele type, 3627727272 allele type, 36277269595 7272 allele type, 362769595 276972 type, allele type, 3627699 type, allele, The rs72692735 allele type is A, rs72692736 allele type is T.

Further, it is preferable that the sample to be tested is derived from blood, urine, saliva, gastric juice, hair or biopsy of the subject to be tested.

The invention finally provides a method for detecting the genotype of the SNP locus related to nicotine dependence, which comprises the following steps:

(1) using human whole genome DNA to be detected as a template, and respectively adopting 30 pairs of primers shown by SEQ ID NO.1-60 to carry out PCR amplification to obtain amplified fragments;

(2) and carrying out agarose gel electrophoresis on the amplified fragment to separate a target band, cutting the gel to recover a product, and then judging the genotype of the nicotine-dependent SNP locus by using a sanger sequencing method.

Further, preferably, the PCR amplification system is:

1 μ L of Taq DNA polymerase, 10 pmol/. mu.L of forward primer 0.6 μ L, 10 pmol/. mu.L of reverse primer 0.6 μ L, 2 μ L of template DNA, 2 μ L of 10 XBuffer, 2.5mM of dNTP 1.6mL, ddH₂O12.2. mu.L, 20. mu.L in total.

Further, preferably, the PCR amplification procedure is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ or annealing at the annealing temperature in Table 1 for 45s, and extension at 72 ℃ for 45s for 45 cycles; final extension at 72 deg.C for 5min, and storage at 4 deg.C.

The invention does not limit the concrete structure of the detection device, for example, the detection device comprises a detection unit and a detection unit, wherein the detection unit is used for detecting the allelic condition of a plurality of single nucleotide polymorphism sites carried by an individual to be detected to obtain a detection result; the device also comprises a data analysis unit and a processing unit for analyzing and processing the detection result of the detection unit to obtain the susceptibility of the individual to be detected to the nicotine dependence.

The present invention provides a series of Single Nucleotide Polymorphisms (SNPs) that are associated with risk of nicotine dependence. The invention carries out gene typing and phenotype association analysis in more than 1500 Chinese population samples to obtain 30 SNPs related to the nicotine dependence of Chinese Han population. The physical position of the SNP site is determined according to the reference genome GRCh37/hg19, and the source of the number of the SNP is dbSNP 151. The SNP site is located in the section from 8561542 to 9916353 of chromosome 9 of the human genome and in the intron section of the PTPRD (receptor type protein tyrosine phosphatase D) gene.

The plurality of single nucleotide polymorphism sites comprises: rs71507664, rs35841018, rs115729775, rs77835962, rs35831393, rs71497118, rs71492010, rs76717037, rs12347862, rs1323791, rs10816208, rs10816209, rs10816210, rs10978037, rs10978038, rs10978039, rs1408117, rs1408118, rs10816211, rs10816212, rs10816213, rs10978042, rs10978043, rs12380595, rs72692730, rs10978078, rs72692731, rs72692733, rs72692735 and rs 72276936. Through these single nucleotide polymorphism sites, association analysis of nicotine dependence can be performed.

Meanwhile, the relevant genotypes of the sites are found to be: the rs71507664 allele type is A, rs35841018 allele type, the T, rs115729775 allele type is A, rs77835962 allele type, the G, rs35831393 allele type is C, rs71497118 allele type, the C, rs71492010 allele type is A, rs76717037 allele type, the G, rs12347862 allele type is C, rs1323791 allele type, the G, rs 16208 allele type is C, rs10816209 allele type, the G, rs 16210 allele type is C, rs10978037 allele type, the A, rs10978038 allele type is A, rs10978039 allele type, the 361401408117 allele type is 361401401401401401408118 allele type is C, rs 16211 allele type, the C, rs 7272727272727272699 allele type is 3616213 allele type, the C, rs allele type is 3678042 allele type, the 362769595 2769595 279 allele type, the 362769595 279 allele type, the 3627729 allele type, the 36277269595 7272 allele type, the 36277272729 allele type, the 362769595 729 allele type, the 362769595 729 type, the allele type, the, The rs72692736 allele type is T.

The SNP locus related to the invention is found in an intron region of a PTPRD gene by annotation.

The present invention relates to the provision of primer pairs for amplifying the genotypes of the above loci, as shown in Table 1.

TABLE 1

Compared with the prior art, the invention has the beneficial effects that:

(1) the 30 SNP loci provided by the invention are obtained based on the correlation analysis of the whole genome sequencing data of Chinese population, have stronger correlation with the nicotine dependence susceptibility of the Chinese population, and can effectively improve the detection efficiency;

(2) the primer pair for amplifying the SNP loci developed by the invention has strong specificity, high sensitivity, better applicability for different individuals, easy understanding of the whole identification process, simple and convenient operation and accurate and reliable result, and can be widely applied to identification of the SNP loci in individuals of Chinese population;

(3) the invention provides a method for identifying the SNP locus allelic gene type, which is simple and convenient to operate and has accurate and reliable results.

Drawings

FIG. 1 shows haplotype blocks (haplotypeblock) of SNP sites on the PTPRD gene detected by Haploview software.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.