SNP (Single Nucleotide Polymorphism) marker for detecting cervical lesion caused by HPV (Human Papilloma Virus) infection, kit and application

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker for detecting cervical lesions caused by HPV (Human Papilloma Virus) infection, a kit and application, belongs to the technical field of biological medicines, and aims to provide the SNP marker for detecting the occurrence risk of the cervical lesions caused by HPV infection, and the SNP marker comprises a TLR4 gene SNP site rs11536889. The core of the invention integrates PCR amplification and DNA sequencing technologies, can effectively detect the correlation between the TLR4 gene polymorphism and the cervical lesion occurrence risk, can accurately evaluate the susceptible risk of the cervical lesion, and provides a basis for individualized diagnosis and treatment of clinical medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a SNP marker, a kit and an application thereof for detecting cervical lesions caused by HPV infection. Background Art

[0002] Cervical cancer develops from precancerous lesions over a long period of time, that is, from human papillomavirus (HPV) infection through low-grade cervical intraepithelial lesions (LSIL) and high-grade cervical intraepithelial lesions (HSIL) to invasive cervical cancer. Persistent infection with high-risk HPV (HR-HPV) is a clear cause of cervical lesions, and viral DNA can be detected in more than 90% of squamous cervical cancers. Today, HR-HPV typing has become an important method for screening cervical lesions. Liquid-based cytology (TCT), which has evolved from traditional Pap smears, is also widely used in clinical practice. It is a routine item for the first stage of screening for cervical lesions and cervical cancer. Pathological tissue biopsy is the gold standard for diagnosis. If early diagnosis can be made at the precancerous lesion stage and early intervention treatment can be carried out, it is possible to prevent the occurrence of cervical cancer and improve the cure rate and survival rate of cervical cancer.

[0003] TLR4 is one of the important members of the Toll-like receptor (TLRs) family. It can recognize pathogen-associated molecular patterns (PAMPs) and damage molecular patterns (DAMPs), stimulate downstream signaling pathways, regulate the host's innate and adaptive immune responses, and play an important role in anti-infection and tumor immunity. In addition, the TLR4 signaling pathway molecule NF-κB controls the expression of a variety of inflammatory factors, and chronic inflammation is closely related to the occurrence and development of tumors. Therefore, TLRs may be associated with cancers such as cervical cancer.

[0004] Single nucleotide polymorphisms (SNPs) are third-generation genetic markers, which refer to single nucleotide mutations with a frequency greater than 1% in the population and are the most common heritable human variation. The occurrence of cervical lesions is also related to individual genetics. Exploring susceptibility genes related to cervical lesions can be used as an important molecular marker to assess the risk of the disease, which has positive significance for the prevention and diagnosis of cervical lesions.

[0005] There are SNP markers in the prior art that assist in the diagnosis of cervical cancer. Although they also use SNP markers to assist in the diagnosis of cervical cancer, they have the following problems: First: There is no SNP marker for the TLR4 gene. The base variation at the rs11536889 site of the TLR4 gene, as one of the biomarkers, can be used to identify carriers of cervical lesion susceptibility sites, detect human susceptibility to cervical lesions, screen individuals susceptible to cervical lesions, and assess the risk of cervical lesions.

[0006] Second: There are limitations in terms of accuracy. The present invention uses first-generation sequencing technology, which has the advantage of high accuracy.

[0007] Therefore, it is necessary to develop a SNP marker, a kit and an application for detecting cervical lesions caused by HPV infection to solve the above problems existing in the prior art. Summary of the invention

[0008] The purpose of the present invention is to provide a SNP marker, a kit and an application for detecting cervical lesions caused by HPV infection, to assess the risk of cervical lesions by screening individuals carrying susceptible genes, and to provide a new means for the screening and diagnosis of cervical lesions.

[0009] The technical solution of the present invention is: A SNP marker for detecting cervical lesions caused by HPV infection, including a SNP site related to cervical lesions caused by HPV infection, characterized in that the SNP site is located on human chromosome 9. The specific SNP site is TLR4 gene SNP site rs11536889.

[0010] The material for detecting SNP markers related to cervical lesions caused by HPV infection includes specific amplification primers for the SNP site rs11536889.

[0011] The specific amplification primers for the SNP site rs11536889 include an upstream primer and a downstream primer Upstream primer: 5'-GATGGACCTCTGAATCTCTT-3' Downstream primer: 5′-TCATAACGGCTACACCATT-3′.

[0012] The invention discloses a method for preparing a product for diagnosing the risk of cervical lesions by using a substance for detecting SNP markers related to cervical lesions caused by HPV infection; the method comprises a specific amplification primer for detecting the SNP site rs11536889.

[0013] The use of the SNP marker for detecting cervical lesions caused by HPV infection in the preparation of a diagnostic kit for detecting cervical lesions caused by HPV infection comprises the following steps: 1. Amplify the partial fragment of the target gene region of SNP site rs11536889 by PCR; 2. Determine SNP typing through sequence analyzer analysis.

[0014] The advantages of the present invention are: The SNP marker, kit and application for detecting cervical lesions caused by HPV infection are used to analyze the genotype of the SNP site rs11536889 on the human TLR4 gene, which can be used to assess the risk of an individual developing cervical lesions, so as to facilitate the early diagnosis and treatment of cervical lesions. The use of specific primer pairs has the advantage of high specificity. The present invention only requires a small amount of DNA sample to measure the polymorphism of the gene. It explores the genetic molecular mechanism of cervical lesions from the perspective of immunogenetics, and provides a new direction for exploring drug targets for early prevention and treatment of cervical lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the agarose gel electrophoresis diagram of the PCR amplification product of the TLR4 gene SNP site rs11536889.

[0016] Figure 2 This is the GG genotype sequencing result of the TLR4 gene SNP site rs11536889.

[0017] Figure 3 This is the CC genotype sequencing result of the TLR4 gene SNP site rs11536889. DETAILED DESCRIPTION

[0018] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0019] In this study, we obtained genetic polymorphism sites associated with susceptibility to cervical lesions caused by HPV infection in case-control analysis, including the TLR4 gene SNP site rs11536889. By detecting the rs11536889 site, we can assess the susceptibility risk of cervical lesions caused by HPV infection, screen high-risk populations, facilitate early diagnosis of the disease, and provide new directions for the development of drug targets for the treatment of cervical lesions.

[0020] The TLR4 gene SNP marker is closely related to the susceptibility to cervical lesions caused by HPV infection. When the genotype of the TLR4 gene SNP site rs11536889 is CC, the subject is susceptible to cervical lesions caused by HPV infection, or has a poor prognosis. Otherwise, the subject is not susceptible to cervical lesions caused by HPV infection and has a good prognosis.

[0021] Single Nucleotide Polymorphisms (SNPs): Single Nucleotide Polymorphisms are the most common DNA sequence variations in the human genome and have gained increasing importance in various biological and biomedical applications. SNPs can be used to explore the evolutionary history of human populations and analyze forensic samples, and therefore play an important role in genetics. Immunogenetics uses these DNA variations to elucidate the underlying genetic factors that underlie the risk of different diseases. The present invention relates to the identification of SNPs for specific diseases, which are specifically identified as SNP sites associated with cervical lesions. The identification of SNPs involved in cervical lesions helps to better understand the disease process and improve diagnostic and therapeutic reagents.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] Implementation Cases 1.SNP site According to case-control association studies, the SNP site rs11536889 of the TLR gene on chromosome 9 was significantly associated with cervical lesions caused by HPV infection. rs11536889 is located in the 3' untranslated region (3' UTR) of the gene.

[0024] The study included 208 patients with cervical lesions caused by HPV infection as the case group, and 204 patients with HPV infection without cervical lesions as the control group. Clinical data and cervical exfoliated cells were collected, and the above-mentioned SNP sites were genotyped.

[0025] 2. Collection of cervical exfoliated cell samples and extraction of genomic DNA The genomic DNA of cervical exfoliated cells was extracted using the magnetic bead method nucleic acid pre-packaging rapid extraction kit provided by Guangzhou Meiji Biotechnology Co., Ltd. The operation steps were in accordance with the kit instructions.

[0026] 3. Primer Design The base sequence near the TLR4 rs11536889 site was obtained from NCBI, and primers were designed using Primer software. The designed primers were compared with the human whole genome sequence using the NCBI BLAST program to verify the specificity of the primers. Highly specific primers were selected and the primer sequences were submitted to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. The primer sequences are as follows: Upstream primer: 5'-GATGGACCTCTGAATCTCTT-3' Downstream primer: 5′-TCATAACGGCTACACCATT-3′.

[0027] 4. PCR amplification reaction system and conditions The PCR amplification system was: 10×Buffer 2.5μL, dNTP 2μL, 5U / μL Taq enzyme 0.125μL, 20μM upstream primer 1.25μL, 20μM downstream primer 1.25μL, genomic DNA 2μL, ddH2O 15.875μL.

[0028] The PCR reaction conditions were 95°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 57.4°C annealing for 30 s, 72°C extension for 60 s, 35 cycles, and finally storage at 4°C.

[0029] 5. Sequencing to determine genotype PCR products were separated and detected by 2.5% agarose electrophoresis. The results are shown in Figure 1 The amplified band was consistent with the target size (441 bp), and there were no impurities and primer dimers, indicating that the amplification primers for the TLR4 gene SNP site rs11536889 had high amplification efficiency and good specificity. After observing the PCR amplification product, it was sent to Shanghai Biotech Co., Ltd. for sequencing verification. The results are shown in Figure 2 and Figure 3 shown.

[0030] The SNP site rs11536889 of the above-mentioned cases and controls was genotyped, and statistical analysis was performed to verify the risk site. The specific implementation is as follows: SPSS 26.0 software was used for statistical analysis. The SNP genotypes in the case group and the control group were analyzed using the Logistic regression model, and the odds ratio (OR) and 95% confidence interval (CI) were calculated to represent the risk coefficient. The χ2 test was used for judgment. P < 0.05 was considered to be significantly different.

[0031] Relationship between the genotype of TLR4 gene SNP locus rs11536889 and cervical lesions caused by HPV infection

[0032] In the table, GG represents the wild genotype, CC represents the mutant genotype, and GC represents the mutant heterozygous genotype. The experiment proved that in the case group consisting of 208 patients with cervical lesions caused by HPV infection and the control group consisting of 204 patients with HPV infection without cervical lesions, the difference in gene distribution frequency of rs11536889 polymorphism locus was statistically significant, with a P value of 0.034. Logistic regression analysis found that compared with the wild genotype GG, the mutant genotype CC increased the risk of cervical lesions, with an odds ratio (OR) of 2.548. In summary, it shows that the risk of cervical lesions in people carrying the rs11536889 mutant genotype is higher than that in people carrying the wild genotype. The rs11536889 genotype can be identified to screen susceptible individuals and assess the risk of cervical lesions.

[0033] The SNP marker, kit and application for detecting cervical lesions caused by HPV infection are used to analyze the genotype of the SNP site rs11536889 on the human TLR4 gene, which can be used to assess the risk of an individual developing cervical lesions, so as to facilitate the early diagnosis and treatment of cervical lesions. The use of specific primer pairs has the advantage of high specificity. The present invention only requires a small amount of DNA sample to measure the polymorphism of the gene. It explores the genetic molecular mechanism of cervical lesions from the perspective of immunogenetics, and provides a new direction for exploring drug targets for early prevention and treatment of cervical lesions.

Claims

1. A SNP marker for detecting cervical lesions caused by HPV infection, comprising SNP sites related to cervical lesions caused by HPV infection, characterized in that: The SNP site is located on human chromosome 9; the specific SNP site is the TLR4 gene SNP site rs11536889.

2. The SNP marker for detecting cervical lesions caused by HPV infection according to claim 1, characterized in that: The material for detecting SNP markers related to cervical lesions caused by HPV infection includes specific amplification primers for the SNP site rs11536889.

3. The SNP marker for detecting cervical lesions caused by HPV infection according to claim 2, characterized in that: The specific amplification primers for the SNP site rs11536889 include an upstream primer and a downstream primer.

4. The SNP marker for detecting cervical lesions caused by HPV infection according to claim 3, characterized in that: The upstream primer is: 5'-GATGGACCTCTGAATCTCTT-3'; the downstream primer is: 5'-TCATAACGGCTACACCATT-3'.

5. The SNP marker for detecting cervical lesions caused by HPV infection according to claim 1, characterized in that: Use of the substance for detecting SNP markers related to cervical lesions caused by HPV infection in the preparation of a product for diagnosing the risk of cervical lesions; Includes specific amplification primers for detecting the SNP site rs11536889.

6. The SNP marker for detecting cervical lesions caused by HPV infection according to claim 1, characterized in that: The use of the SNP marker for detecting cervical lesions caused by HPV infection in the preparation of a diagnostic kit for detecting cervical lesions caused by HPV infection comprises the following steps: (1) Amplify a partial fragment of the target gene region of the SNP site rs11536889 by PCR; (2) Determine SNP typing through sequence analyzer analysis.

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