CN107557498A - RelA genes rs11820062SNP is preparing the application in detecting hepatitis C neurological susceptibility product - Google Patents

Abstract

The invention discloses a kind of RelA gene rs11820062 SNPs of NF κ B paths in human genome（single nucleotide polymorphism,SNP）Or application of the genotype in detection or the product of examination hepatitis C neurological susceptibility product or the SNP related to hepatitis C is prepared；In actual applications, rs11820062 polymorphism will can be detected（That is allele）Or genotype material and other materials（Such as detect the other SNPs or genotype material related to hepatitis C）It is united the product for preparing examination hepatitis C susceptible person.

Description

RelA gene rs11820062 SNP in the preparation and detection of hepatitis C susceptibility products Applications

technical field

The invention relates to the field of biomedicine, in particular to the application of the rs11820062 single nucleotide polymorphism (single nucleotide polymorphism, SNP) of the RelA gene rs11820062 in the NF-κB pathway in the human genome in the preparation and detection of hepatitis C susceptibility products.

Background technique

SNP refers to a change in nucleic acid sequence caused by a single nucleotide mutation. There are two alleles at a SNP locus. In the human genome, there is one SNP in every 100-300 nucleotides on average. SNPs are the most common type of genetic variation in humans. SNPs are distributed in both coding and non-coding regions of the human genome, and most SNPs exist in non-coding regions. SNPs in the coding region have non-synonymous mutations and synonymous mutations. Non-synonymous SNPs can change the amino acid sequence of proteins, while synonymous mutations do not affect protein coding. SNPs in non-coding regions may affect gene splicing, transcription factor binding, mRNA degradation, etc. The distribution of human SNPs is different, which leads to the diversity of chromosomal genomes of different races and populations, and also leads to differences in human susceptibility to diseases, disease progression, outcome, and drug treatment effects. At present, SNPs have been regarded as a kind of biomarkers and are widely used to study the relationship between genetic variation and human diseases.

Viral hepatitis C is a type of viral hepatitis caused by infection with the hepatitis C virus (HCV). There are 185 million HCV infected people in the world, and the HCV infection rate in China is about 1.6%. Blood transfusion, injection drug use, organ transplantation and hemodialysis have become the main routes of HCV transmission in China. Acute HCV infection can have two distinct outcomes: viral self-limited clearance and persistent infection. Self-limited clearers refer to those who are infected with HCV, and the body automatically clears HCV within 12 months after infection without treatment, so as to avoid persistent infection, manifested as HCV RNA negative and HCV antibody positive. Persistent infection means that after HCV infects the human body, the body cannot clear HCV within 12 months of infection without treatment, and it is manifested as HCV RNA positive and HCV antibody positive. More than 70% of HCV-infected patients may develop persistent infection, and some of them may develop liver cirrhosis and hepatocellular carcinoma (HCC).

The disease progression and outcome of HCV-infected patients can be affected by many factors, including viral, host immune and genetic factors. In the past few years, in many large-scale clinical trials around the world, the new direct-acting antiviral agents (DAAs) have significantly increased the cure rate of chronic hepatitis C to more than 95%, and have become the traditional medicine of polyethylene glycol. A better choice for interferon + ribavirin treatment. However, with the application of DAAs, new problems continue to emerge: HBV/HCV co-infected patients using DAA drugs can cause HBV reactivation; Can not reduce the incidence of HCC; DAAs treatment can also lead to the occurrence of drug resistance, the currently confirmed drug resistance associated mutation (resistance associated variants, RAVs) sites include NS3/4A target-related, NS5A target-related and NS5B target Related dozens of RAVs.

Considering the primary issue in preventing HCV transmission is screening and identifying susceptible populations. Therefore, research on the relationship between genetic background and HCV infection in the Chinese population is of great importance for in-depth exploration of the mechanism of genetic factors in the progression of hepatitis C, and for providing effective targets, tools and theoretical basis for timely and effective prevention and control of hepatitis C epidemic. Practical significance.

NF-κB is a nuclear transcription factor, which can combine with specific nucleotide sequences in the promoter region of some genes to initiate gene transcription, regulate cell proliferation, apoptosis, inflammatory process and immune response. NF-κB can be activated by many stimuli such as bacterial/viral antigens and cytokines, and then regulate the expression of more than 150 genes. NF-κB/ReL family members include c-ReL, NF-κB1 (P50), NF-κB2 (P52), ReLA (P65), and ReLB. These proteins have an amino-terminus consisting of about 300 amino acids, called ReL homology region, including DNA binding site, dimerization site and binding site with NF-κB inhibitory protein (IκB). Active DNA-binding NF-κB is a dimer of P50 and P65. ReL protein members can form homologous or heterologous dimers. Different NF-κB/ReL protein dimers have different binding sequences and have their own characteristics. Different dimers can recognize different DNA targets. The ability to express different regulatory genes is improved. NF-κB protein P50/P65, P50/c-ReL, and P65/c-ReL complexes have transcriptional activation, while P50/P50 and P52/P52 homodimers have transcriptional repression.

Existing studies have shown that NF-κB family genes (NF-κB1, NF-κB2, RelA, RelB, ReL) and their inhibitory gene IκB may affect the host's susceptibility to HCV and the antiviral immune response in vivo through related SNPs . In 2014, a study showed that NF-κBε (IkBε) rs2233437-A significantly improved the self-limiting clearance of HCV in the host, suggesting that polymorphisms of genes related to the NF-κB pathway may be associated with the outcome of HCV infection. But so far there are only studies reporting that rs11820062 is associated with susceptibility to chronic kidney disease (O'Brown, Z.K., Van Nostrand, E.L., Higgins, J.P. & Kim, S.K. The inflammatory transcription factors NFκB, STAT1 and STAT3 drive age-associated transcriptional changes in the human kidney.PLoS genetics 11, e1005734 (2015)) and schizophrenia (Hashimoto, R. et al. Variants of the RELA gene are associated with schizophrenia and their startle responses. Neuropsychopharmacology 36, 1921-1931 (2011)), rs230530 and liver cancer (Gao, J. et al. Genetic polymorphism of NFKB1 and NFKBIA genes and liver cancer risk: a nested case–control study in Shanghai, China. BMJ open 4, e004427 (2014).) and alcohol dependence (Edenberg, H.J. et al. Association of NFKB1, which encodes a subunit of the transcription factor NF-κB, with alcohol dependence. Human molecular genetics 17, 963-970 (2008).). However, there is no report on the relationship between the polymorphism of RelA gene rs11820062 single nucleotide site and the susceptibility to hepatitis C infection at home and abroad.

Contents of the invention

The purpose of the present invention is to provide an application of rs11820062 single nucleotide polymorphism of NF-κB pathway RelA gene in the preparation of products for detection or screening of hepatitis C susceptibility.

Specifically, the present invention first provides any of the following purposes:

(1) Application of single nucleotide polymorphism (ie allele) or genotype of RelA gene rs11820062 in human genome in the preparation of products for detection or screening of hepatitis C susceptibility.

(2) Application of single nucleotide polymorphism (ie allele) or genotype of RelA gene rs11820062 in human genome in the preparation of products for detection or screening of single nucleotide polymorphisms related to hepatitis C.

Secondly, the present invention also provides any of the following purposes:

(1) Application of a substance for detecting the single nucleotide polymorphism (ie allele) or genotype of RelA gene rs11820062 in the human genome in the preparation of products for detecting or screening hepatitis C susceptibility.

(2) Substances for detecting single nucleotide polymorphisms (ie alleles) or genotypes of RelA gene rs11820062 in the human genome are used in the preparation of products for detecting or screening single nucleotide polymorphisms related to hepatitis C Applications.

In the present invention, the substances for detecting the single nucleotide polymorphism or genotype of the RelA gene rs11820062 in the human genome include reagents or kits that can be used for detecting genotypes in the prior art, specifically, these substances are conventional in the art The reagents and kits involved in the detection method that can be used to detect the single nucleotide polymorphism of RelA gene rs11820062.

The commonly used detection methods for human genome SNP typing include: single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), enzyme-cut amplified polymorphic sequence (CAPS), and allele-specific PCR. (including the TaqMan-PCR method used in the present invention), detection, direct gene sequencing (Sanger method), DNA chip method, denaturing high performance liquid chromatography (DHPLC) and mass spectrometry, etc. The reagents used by different SNP typing methods are different; while the same type of detection method uses differently designed primers, probes, etc. according to the researcher's different detection purposes, sites and typing effects, to obtain different genotypes. reagents or kits.

In the present invention, the product for detecting or screening hepatitis C susceptibility includes the above-mentioned substance for detecting the single nucleotide polymorphism or genotype of the RelA gene rs11820062 in the human genome, which can be a reagent or a kit, and can also be a reagent or a kit Combination products with instruments, such as products obtained by combining primers and DNA sequencers, and products obtained by combining TaqMan PCR reagents, DNA sequencing reagents and DNA sequencers for detection or screening of hepatitis C susceptibility.

In the present invention, the products for detecting or screening single nucleotide polymorphisms related to hepatitis C also include the above-mentioned substances for detecting the single nucleotide polymorphism or genotype of RelA gene rs11820062 in the human genome, which can be reagents or The test kit can also be a combination product of reagents, kits and instruments, such as the product obtained by combining primers and DNA sequencer, the detection or screening of hepatitis C and hepatitis C obtained by the combination of TaqMan PCR reagent, DNA sequencing reagent and DNA sequencer Related SNP products.

The present invention also provides a TaqMan PCR reagent, which contains PCR primers and TaqMan MGB probes for amplifying genomic DNA fragments including RelA gene rs11820062 in human genome.

Further, in the TaqMan PCR reagent provided by the present invention, the PCR primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the TaqMan MGB probe sequences are shown in SEQ ID NO.3 and SEQ ID NO .4, and the 5' ends of SEQ ID NO.3 and SEQ ID NO.4 are labeled with a fluorescent reporter group, and the 3' ends are labeled with a non-fluorescent quencher group and a DNA minor groove binder MGB group.

In the present invention, rs11820062 is a biallelic polymorphic SNP site on human chromosome 11q13.1, and the variation is conversion (G→A, then C→T on its complementary strand), the rs11820062 gene Genotype refers to genotype GG, GA or AA; said GG is the wild genotype whose rs11820062 site is G, said AA is a homozygous mutant genotype whose rs11820062 site is A, and said GA is a genotype whose rs11820062 site is Heterozygous mutant genotypes of G and A. The detection of the single nucleotide polymorphism or genotype of rs11820062 in the human genome specifically refers to the detection of the nucleotide type of rs11820062. The examples of the description of this application show that the proportion of individuals carrying the homozygous mutant AA genotype of rs11820062 in the HCV infected population (case group) is higher than that of the uninfected HCV population (control group) carrying the homozygous mutant AA genotype Therefore, it is considered that individuals carrying the AA genotype are more susceptible to hepatitis C than those carrying the RelA gene rs11820062GG wild genotype.

Further, the present invention detects the RelA gene rs11820062 single nucleotide polymorphism (i.e. allele) or genotype in the human genome, including TaqMan-PCR reagents, which include amplifying the RelA gene rs11820062 genomic DNA fragments PCR primers and TaqMan probes.

Further, in the present invention, the PCR primer refers to: the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; the TaqMan probe Including: Probe for detecting wild allele G (Probe-G): Label the fluorescent reporter group FAM at the 5' end of the sequence of SEQ ID NO.3, and label the non-fluorescent quenching NFQ group and the DNA minor groove at the 3' end The binder MGB group; and the probe (Probe-A) for detecting the mutant allele A: the fluorescent reporter group VIC is labeled at the 5' end of the SEQ ID NO.4 sequence, and the non-fluorescence quenching NFQ group is labeled at the 3' end group and DNA minor groove binder MGB group.

In the application of the present invention, the hepatitis C mentioned specifically refers to the hepatitis C of the Chinese Han population.

The HCV-infected persons (case group) described in the application of the present invention are the combined population of HCV self-limited clearance and persistent infection, that is, the population without anti-HCV treatment and HCV antibody positive (HCV RNA negative or positive). The HCV-uninfected population (control group) described herein is a non-HCV-infected population with negative HCV antibody and negative HCV RNA.

The 5' end of the TaqMan-MGB probe used in the present invention is connected with a fluorescent reporter group: 6-carboxyfluorescein (FAM) or hexachloro-6-methylfluorescein (VIC), and the 3' end is connected with a non-fluorescent The quencher group (non-fluorescent quencher, NFQ) itself does not produce fluorescence, but has the effect of quenching the fluorescence emitted by the fluorescent reporter group. The probe is also connected with a DNA minor groove binder (MGB) modification group, which can be embedded in the minor groove in the DNA double helix structure to form a non-covalent bond and improve the stability of the hybrid chain. TaqMan-MGB probes have higher sensitivity and specificity than conventional TaqMan probes, and are effective tools for detecting single-base mutations.

At the beginning of PCR amplification, the probe is intact, the fluorescent reporter group is very close to the quencher group, the fluorescent signal is absorbed by the quencher group, and no fluorescence intensity can be detected at this time. When the product to be tested is amplified by PCR, the Taqman probe first binds to the complementary part of the DNA template sequence, and as the extension reaction proceeds, the TaqDNA polymerase moves along the template chain, and when it reaches the position of the fluorescent reporter group, it exerts a 5' →The 3' cleavage activity cleaves the probe, at this time the fluorescent reporter group is separated from the quencher group, resulting in a detectable fluorescent signal. The synthesis of each new PCR strand is accompanied by the release of a fluorescent signal whose intensity is proportional to the amount of PCR product. As the number of amplifications increases, the intensity of the released fluorescent signal increases continuously. The PCR product in the sample well can be genotyped by real-time dynamic detection of the fluorescence intensity emitted in the reaction.

In the present invention, in the case group composed of 1456 HCV infected persons, and in the control group composed of 1125 non-infected HCV populations, the proportion of individuals carrying the AA genotype in the case group population is higher than that of this genotype in the non-infected The proportion in the HCV population (control group). This shows that compared with the wild genotype carrying RelA gene rs11820062GG, individuals carrying the AA genotype are more susceptible to hepatitis C, with a relative risk of 1.531 times, which is statistically significant (P=0.002). In the additive model, dominant model and recessive model, there is still a statistical correlation between the rs11820062A allele and HCV susceptibility, and the relative risk is 1.241 times (P=0.002), 1.354 times (P=0.003) and 1.346 times (P=0.018). In practical applications, the detection of polymorphisms (i.e. alleles) or genotypes of rs11820062 can be combined with other substances (such as detection of other single nucleotide polymorphisms (i.e. alleles) associated with hepatitis C or genotype material) are combined to prepare a product for screening hepatitis C susceptibility.

In one embodiment of the present invention, the genomic DNA fragment including rs11820062 is amplified by the TaqMan-MGB probe method, and the two alleles on the same gene locus are marked with two Taqman fluorescent probes, using the ABI 7900HT type Fluorescent quantitative PCR instrument detects the relative fluorescence intensity of the probe in each sample well on a 384-well plate, interprets the genotyping results with the end-point plate reading program, and determines the genotype according to the type and intensity of the fluorescent signal, which can realize a large number of samples high-throughput allelic typing.

According to the SNPs site to be detected, the present invention designs two different probes and forward and reverse primers respectively, respectively uses FAM and VIC to fluorescently label the probes, and analyzes its specificity to ensure that each primer is compatible with the database. Other human genes have no homology. When performing TaqMan-PCR reaction, if the two bases of the two chromosomes are the same and only emit one kind of fluorescence, it can be judged as a homozygous genotype (AA or GG), and the homozygous type can be distinguished according to different fluorescent signals Gene type: wild type (GG) or homozygous mutant (AA); when the bases of the two polymorphic sites are different, there will be two fluorescent signals, indicating that the genotype of the sample to be tested is a heterozygous mutant (AG). The test results are shown in Figure 1.

The present invention finds that the RelA gene rs11820062 is a single nucleotide polymorphism associated with the susceptibility to hepatitis C in a sample (1456 HCV-infected and 1125 controls) from the Chinese Han population, and the single nucleotide polymorphism of rs11820062 can be detected nucleotide polymorphism (i.e. allelic) or genotype material in combination with other material (eg detection of other HCV-associated SNPs (i.e. allele) or genotype material) Products for the screening of persons susceptible to hepatitis C are prepared together.

Description of drawings

Figure 1 is a genotyping diagram of the rs11820062 locus of the RelA gene by the TaqMan-MGB probe method.

Figure 2 is a schematic diagram of the secondary structure of the center of gravity of the mRNA near the rs11820062 site at the 5' end of the RelA gene.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only to illustrate the present invention, rather than to limit the scope of the present invention.

The examples of the present invention contain the following materials and reagents, which can be obtained from commercial sources unless otherwise specified. Nucleotide sequences involved in the following examples:

SEQ ID NO.1 (Forward primer sequence of rs11820062 site):

CTTGACTCAGTTTCCCTCCCACAC;

SEQ ID NO.2 (reverse primer sequence of rs11820062 site):

GAGGGAAAACGGGGTAAGGAATC;

SEQ ID NO.3 (rs11820062 site Probe-G): TCCCTCAGTTTTC;

SEQ ID NO.4 (rs11820062 site Probe-A): TCCCTCAATTTTC;

SEQ ID NO.5 (common primer): AGGAAGACTTCCGAGCGGTC;

SEQ ID NO.6 (HCV 1a genotype specific primer): TGCCTGGGGATAGGCTGAC;

SEQ ID NO.7 (HCV 1b genotype specific primer): GAGCCATCCTGCCCACCCCA;

SEQ ID NO.8 (HCV2 genotype-specific primer): CCAAGAGGGACGGGAACCTC;

SEQ ID NO.9 (HCV 3 genotype specific primer): ACCCTCGTTTCCGTACAGAG;

SEQ ID NO.10 (HCV4 genotype-specific primer): GCTGAGCCCAGGACCGGTCG;

SEQ ID NO.11 (forward primer sequence of rs1056890 site): TGGGCCTCAGGAGCCTAG;

SEQ ID NO.12 (reverse primer sequence of rs1056890 site):

ATCAAAAGTTCAGGGGCGCTAG;

SEQ ID NO.13 (rs1056890 site Probe-C): CACCTCCGAGAGC;

SEQ ID NO.14 (rs1056890 site Probe-T): CACCTCTGAGAGCC;

SEQ ID NO.15 (wild type mRNA sequence at rs11820062 site):

CAGAGGGAAGCUGAAUCAGGGCCUGUUGUACUUUCUUAAGGAAAACUGAGGGAGGGCACGCCCCACCUCCCUCCAGAGAGGAAACUGAAUC;

SEQ ID NO.16 (rs11820062 site mutant mRNA sequence):

CAGAGGGAAGCUGAAUCAGGGCCUGUUGUACUUUCUUAAGGAAAAUUGAGGGAGGGCACGCCCCACCUCCCUCCAGAGAGGAAACUGAAUC.

The experimental methods in the following examples are conventional methods unless otherwise specified.

All research subjects in the following examples signed a written informed consent. The protocol of this study was approved by the Ethics Committee of Jiangsu Provincial People’s Hospital, and complied with the terms of the ethical guidelines for human medical research in the newly revised Declaration of Helsinki of the World Medical Association in 2013 (World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013, 310(20): 2191-2194.).

Research objects: the following examples are included in total: (1) 898 HCV persistent infection patients: serum HCV antibody positive (detected with the third generation ELISA kit of Abbott Company), and HCV RNA positive, ALT increased or normal; (2) 558 HCV self-limited clearance subjects: serum HCV antibody positive and HCV RNA negative, ALT elevated or normal; (3) 1125 controls, serum HCV antibody negative and HCV RNA negative. Group (1) and group (2) are collectively referred to as the infection group (case group). The control group was matched with the persistent infection group and the self-limited clearance group in terms of age (<5 years old), gender, and geographic region (urban, rural). Patients co-infected with other hepatitis viruses, HIV or treated with antiviral drugs were excluded. During the study period, all serological results were validated by at least three independent experiments with consecutive 12-month follow-up periods. All subjects were diagnosed by experienced doctors with internationally recognized standards on the basis of clinical and laboratory data.

Example 1 Preparation of substances for detecting the single nucleotide polymorphism or genotype of the RelA gene rs11820062 in the human genome

1. Genomic DNA extraction

(1) Collect 5ml of peripheral venous blood from patients in EDTA anticoagulant tubes, centrifuge at 4000rpm for 10min, separate serum, white blood cells and red blood cells, number them one by one after aliquoting, and freeze them at -80°C for later use.

(2) Genomic DNA was extracted by the phenol-chloroform method: take 3 times the volume of cell lysate and add it to blood cells after centrifugation, shake and mix well, lyse at room temperature for 5 minutes, centrifuge at 4000rpm for 10 minutes, and discard the supernatant.

(3) Observe the color of the precipitate. If the red color is darker, continue to add cell lysate, break and remove red blood cells until the centrifuged precipitate is white or light pink.

(4) Take 1ml of genomic DNA extract and 8μl of proteinase K solution, add to the precipitate obtained in (3), mix thoroughly, and place in a water bath at 37°C overnight.

(5) Take out the centrifuge tube and let it cool down, add 1ml of Tris saturated phenol, press the cap of the tube tightly, turn it upside down for 15min, mix well, and centrifuge at 4000rpm for 10min.

(6) Take the supernatant and transfer it to another clean centrifuge tube, add chloroform: isoamyl alcohol mixture (24:1) equal to the volume of the supernatant, press the tube cap tightly, turn it upside down for 15 minutes, and mix thoroughly. Evenly, centrifuge at 4000rpm for 10min, take the supernatant, and evenly distribute it in 2 clean centrifuge tubes.

(7) Add 1/10 volume of NaAC solution (3mol/L), swirl gently and mix thoroughly. Then add an equal volume of -20°C pre-cooled ice-free ethanol, gently invert several times up and down, white flocculent precipitates can be seen with the naked eye, centrifuge at 8000-10000rpm for 10min, and discard the supernatant.

(8) Add 1ml of ice-free ethanol pre-cooled at -20°C to the white precipitate again, press the tube cap tightly, shake up and down, wash the inner wall of the tube, and the DNA falls into the solution in the tube. Then centrifuge at 12000rpm for 10min, discard the supernatant, and repeat the operation once.

(9) Invert the centrifuge tube and dry it on clean filter paper for 20 minutes, or drain the ethanol with a vacuum desiccator.

(10) Add 100 μl TE buffer solution, overnight at 4°C, after the DNA is completely dissolved, check the numbers one by one, and place in a 4°C refrigerator.

(11) Take 200 μl of DNA solution, dilute it to 100 μl, measure the absorbance value of OD ₂₆₀ and OD ₂₈₀ by UV spectrophotometry, determine the DNA concentration by OD ₂₆₀ , 1 ~ 20 ng/μl meets the experimental requirements; use the ratio of OD ₂₆₀ /OD ₂₈₀ Determine the purity of DNA, between 1.8-1.9 meets the experimental requirements.

(12) According to the measured DNA concentration, take an appropriate amount and dilute it to 50-200ng/μl, mix the DNA thoroughly, and store it in a refrigerator at 4°C for use in SNP genotyping by PCR or Taqman method. The original DNA solution was frozen at -20°C.

2. HCV RNA extraction

(1) Take 300 μl of serum, add 1ml of RNA iso Plus, shake and mix, and let stand at room temperature for 5 minutes.

(2) Add 200 μl of chloroform to the Eppendorf tube, press the cap of the tube tightly with your fingers, shake vigorously for 15 s, so that the liquid in the tube is fully emulsified and no longer separate layers, and stand at room temperature for 5 min again.

(3) Place in a refrigerated high-speed centrifuge, centrifuge at 12,000 rpm for 15 minutes at 4°C.

(4) Absorb the supernatant after centrifugation, avoid sucking into the white layer in the middle, add it into a new clean Eppendorf tube, then add an equal volume of isopropanol, mix it upside down, and let it stand at room temperature for 10 minutes.

(5) Place in a refrigerated high-speed centrifuge, centrifuge at 12000 rpm for 10 min at 4°C.

(6) Aspirate and discard the supernatant, slowly add 1ml of 75% ethanol along the wall of the Eppendorf tube, and wash the tube wall by inverting gently.

(7) Place in a refrigerated high-speed centrifuge, centrifuge at 12,000 rpm at 4°C for 5 minutes, and suck up the supernatant.

(8) Let stand at room temperature for 10 minutes, dry the precipitate, add appropriate amount of water (RNase-free), gently blow with a pipette to make the precipitate fully, and freeze at -80°C.

3. HCV RNA amplification

Take 5 μl of HCV RNA obtained in the previous step as a template, pre-denature at 70°C for 10 minutes, and ice-bath for 5 minutes, then add the following reagents in sequence: 5×buffer 4 μl, 10 mmol/L dNTP 1 μl, 20 U/μl RNase 0.5 μl, 50 pmol/μl downstream primer 0.5 μl, 2 μl of 0.1 mol/L dithiothreitol (DTT), 1 μl of M-MLV reverse transcriptase, 11 μl of sterilized water added with diethylpyrocarbonate (DEPC), the total volume of the reaction system was 20 μl. Reverse transcription at 37°C for 1 hour, inactivation at 95°C for 5 minutes, and rapid ice bath for 5 minutes.

4. Quantification of HCV RNA

HCV RNA in blood samples was quantified by the Cobas TaqMan HCV Test kit from Roche, USA, and the specific operation steps were carried out according to the instructions of the kit.

5. HCV genotyping

(1) Genotyping of HCV RNA positive patients:

Take 5 μl of the above-mentioned amplification products and add them to 5 Eppendorf tubes, each containing 0.5 μl of the reaction mixture of common primers (nucleotide sequence as shown in SEQ ID NO. , the total reaction system is 50 μl.

Nested PCR amplification conditions: pre-denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, and 72°C for 10 min. 5 μl of the amplified product was subjected to agarose gel electrophoresis, and the HCV genotype was determined according to the size of the fragment.

Table 1 HCV genotype detection primers

(2) Genotyping of HCV RNA-negative patients

Using the Murex HCV Serotyping 1-6 Assay ELISA kit, HCV genotyping was performed on patients with HCV RNA-negative self-limited clearance by ELISA method based on HCV virus type-specific antibodies (Bhattacherjee V, Prescott L, Pike I, et al .Use of NS-4 peptides to identify type-specific antibody to hepatitis C virus genotypes 1,2,3,4,5and 6[J].Journal of generalvirology,1995,76(7):1737-1748.).

The detection principle and brief steps are as follows: 2 highly variable polypeptide sequences in the NS4 region of the HCV non-structural region have linear antigenic epitopes specific to HCV 1-6 genotypes, and artificially synthesize 1-6 specific polypeptide antigens accordingly. Coated microplates. Add neutralizing antigen and serum samples sequentially. There are 7 different types of neutralizing antigens, one contains all 6 types of antigens, and each of the others contains only 5 specific antigens from types 1 to 6, and lacks one of the types of antigens from types 1 to 6 in sequence. And antibodies are added in the order of microplate F to A, and each row is added sequentially. If the specimen contains type 1 antibody, it cannot be neutralized by the neutralizing antigen that lacks type 1 antigen, but binds to the type 1 antigen coated on the microwell plate .

After the mixture was incubated at 37°C for 1 hour, it was washed to remove unbound substances, and the captured specific antibody was then combined with HRP-labeled secondary antibody IgG. After 1 hour at 37°C, a chromogenic substrate was added and measured at 450nm with a microplate reader. OD value. Set the control H well without adding neutralizing antigen, and the control G well adding all 6 types of neutralizing antigen, and confirm the antibody typing results with "OD _H /OD _G ≥0.1" and "OD _Sample /OD _G ≥0.4" , the specific operation and result judgment shall be carried out according to the instructions.

6. Screening of SNP candidate sites

Download the SNP database of Chinese Beijing population (CHB) in HapMap (http://www.hapmap.org), import HaploView software to select tag SNP (Tag SNP): the parameter is set so that the correlation coefficient r ² is greater than 0.8; in the Chinese population has a high frequency, that is, the minimum allele frequency (minor allele frequency, MAF) is greater than 0.05. Considering that the adjacent sequences, especially the sequences near the upstream promoter region, may have a regulatory effect on the gene, each candidate gene, including the upstream 2000bp and downstream 2000bp sequences of its transcription start point, was included in the analysis. In addition, combined with relevant literature, select Tag SNP sites that may affect gene function. According to the above principles, two candidate sites were selected in this embodiment: rs1056890 and rs11820062.

Rs11820062 is located at the intron 1 or 5' end of the RelA gene (derived from two different graph pipelines in NCBI dbSNP: https://www.ncbi.nlm.nih.gov/snp/ ), and the RelA gene is located on the human chromosome 11q13.1, which contains 11 exons and 10 introns. rs1056890 is located in the 3'-UTR region of the NF-κB2 gene, which is located on human chromosome 10q24.32 and contains 25 exons and 24 introns.

7. SNP locus genotyping

(1) According to the SNPs site of human genome rs11820062, design forward primer (as shown in SEQ ID NO.1) and reverse primer (as shown in SEQ ID NO.2) and two different probes respectively, namely The probes are fluorescently labeled with FAM and VIC respectively (the probe for detecting the wild-type allele G: the nucleotide sequence shown in SEQ ID NO.3, and the 5' end is connected with a fluorescent reporter group FAM, and the 3' End-labeled non-fluorescence quenching NFQ group and DNA minor groove binder MGB; probe for detecting mutant allele A: the nucleotide sequence shown in SEQ ID NO.4, and the 5' end is connected with a fluorescent reporter The group VIC, the 3' end labeled non-fluorescence quenching NFQ group and the DNA minor groove binder MGB, and analyzed their specificity to ensure that each primer has no homology with other human genes in the database.

According to the SNPs site of human genome rs1056890, a forward primer (as shown in SEQ ID NO.11) and a reverse primer (as shown in SEQ ID NO.12) and two different probes were designed respectively, that is, FAM and VIC to fluorescently label the probe (probe for detecting wild-type allele C: the nucleotide sequence shown in SEQ ID NO.13, and the 5' end is connected with a fluorescent reporter group FAM, and the 3' end is labeled with non- Fluorescence quenching NFQ group and DNA minor groove binder MGB; probe for detecting mutant allele T: the nucleotide sequence shown in SEQ ID NO.14, and the 5' end is connected with a fluorescent reporter group VIC, The 3' end is labeled with non-fluorescence quenching NFQ group and DNA minor groove binder MGB, and its specificity is analyzed to ensure that each primer has no homology with other human genes in the database.

(2) The DNA samples to be tested were uniformly diluted to 10 ng/μl, and distributed to 96-well plates for subsequent loading of the sample gun. Centrifuge the dry powder of primers and probes at 12000rpm for 5min. After centrifugation, gently open the tube cap to prevent the dry powder from spraying out after opening the cap. Then add an appropriate amount of sterile double-distilled water to dilute the probe/primer to the specified concentration, shake well to mix.

(3) Centrifuge the probe, primer, and PCR Master Mix solution at 12,000 rpm for 30 seconds, and mix them separately. The DNA samples distributed to the 96-well plate were also centrifuged for 1 min in a centrifuge dedicated to the micro-well plate to mix well. Then prepare the PCR system as shown in Table 2:

Table 2 TaqmanPCR system preparation

The above-mentioned system in Table 2 is the material for detecting the single nucleotide polymorphism or genotype of the RelA gene rs11820062 in the human genome; the above-mentioned material obtained in this example is a genomic DNA fragment including the amplified RelA gene rs11820062 site As for the TaqMan PCR reagents of PCR primers and TaqMan MGB probes, other conventional reagents or kits for detecting SNP genotypes in the art can also be used in specific implementations.

Example 2 Application of RelA gene rs11820062 single nucleotide polymorphism or genotype

1. Detection steps

The substance obtained in Example 1 for detecting the single nucleotide polymorphism or genotype of the RelA gene rs11820062 in the human genome (that is, the system solution prepared according to the system in Table 2) was divided into 8 sterile PCR tubes. Adjust the volume of the discharge gun to 5 μl, take the system solution and add it to a 384-well plate, and finally add 1 μl/well of the DNA sample. Set 2 blank controls (sterile double distilled water) and 2 known positive controls on each plate to control reagents, system contamination and test results.

(1) Seal the 384-well plate containing the sample with a special Taqman PCR sealing film, and centrifuge for 1 min. Install the 7900HT fluorescent quantitative PCR instrument and set the PCR reaction conditions: pre-denaturation at 50°C for 5 minutes, initial denaturation at 95°C for 10 minutes, annealing at 95°C for 30 seconds, extension at 60°C for 30 seconds, 35 cycles.

(2) After the PCR reaction, the genotyping results were read out on the 7900HT fluorescent quantitative PCR instrument with the Sequence Detection System software (SDS, version 2.3) of Applied Biosystems.

The genotyping success rate for loci with 2 SNPs is approximately 95%.

When performing TaqMan-PCR reaction, if the two polymorphic sites have the same base and only emit one kind of fluorescence, it can be judged as a homozygous genotype, and the type of homozygous gene can be distinguished according to different fluorescent signals: wild gene type or homozygous mutant type; when the bases of the two polymorphic sites are different, there will be two fluorescent signals, indicating that the genotype of the SNPs carried by the sample to be tested is a heterozygous mutant type. The genotype detection results of rs11820062 are shown in Figure 1. In Figure 1, GG represents the wild GG genotype, GA represents the heterozygous mutant GA genotype, and AA represents the homozygous mutant AA genotype.

2. Quality control

Interviews were recorded using structured standardized questionnaires for all study participants. The questionnaire collected the main risk factors of HCV infection reported in the literature, including gender, age, region, history of HCV infection, and exposure history of environmental risk factors for hepatitis C. This study formulated an epidemiological survey work manual and standardized operating procedures for experimental testing, and uniformly trained all staff to ensure the quality of survey data and laboratory measurements in this study. All the questionnaires and data were coded by the staff, and the two entered the computer independently, and the database was established after the two sides reviewed it. All test results were interpreted by two researchers in a blinded manner, that is, the two investigators did not know the clinical and other data of the patients from which the samples were tested. Repeat the experiment on samples with unsuccessful detection or questionable interpretation until consistent results are obtained. Each group randomly selected 5% of the samples to repeat the test, and the consistency rate was 100%.

3. Statistical analysis

Two staff members used EpiData 3.1 software to double-track the entry of questionnaire data and test results. After logical checking and checking, a database was established, and SPSS software (version 21.0) and Stata (version 13) were used for further analysis. Quantitative variables were expressed as mean ± standard deviation (SD), or median (interquartile range (IQR)). Between the case group and the control group, the differences in demographic characteristics, biological indicators and allele distribution frequency of HCV patients were analyzed by χ2 test, One-Way ANOVA or non-parametric Kruskal-Wallis test. Goodness-of-fit Pearson's χ2 test was used to assess whether the genotype distribution of the control group was in Hardy-Weinberg equilibrium. Multivariate Logistic regression analysis was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) to analyze the high risk factors of HCV infection. Stratified analysis was used to control for the influence of confounding factors on the statistical results. Multiple comparisons were corrected by the Bonferroni method, which is the most conservative, and a two-sided P value <0.025 was considered statistically significant. In non-multiple comparisons, a two-sided P value < 0.05 was considered statistically significant.

4. Socio-demographic and clinical characteristics of the research subjects

The sociodemographic and clinical characteristics of all study subjects are shown in Table 3. The persistent infection group included 874 patients (341 males, 533 females, mean age 49.73±10.96 years). The self-limited viral clearance group included 557 people (225 males, 332 females, mean age 49.01±13.54 years). The healthy control group included 1124 uninfected individuals (422 males, 702 females, mean age 49.54±14.48 years). There was no significant difference in age and gender among the three groups (P>0.05), but there were significant differences between the three groups in ALT level, AST level, infection route and the control group (P<0.001), and the persistent infection group and self-limited clearance There were significant differences in the infection virus types between the two groups (P<0.001).

Table 3 Demographic and clinical characteristics of HCV persistent infection group, self-limited clearance group and control group

Remarks: Group A: Control group (uninfected group), Group B: Self-limited clearance group, Group C: Persistent infection group, Group (B+C): Infected group (case group). mean: mean; SD: standard deviation; ALT: alanine aminotransferase; AST: aspartate aminotransferase; IQR: interquartile range; aOne-Way ANOVA; bχ2- test; cKruskal–Wallis test.

5. Hardy-Weinberg genetic balance test

For the two SNP sites (rs1056890 and rs11820062) involved in this study, the uninfected group was used as the control group, rs1056890: P=0.834; rs11820062: P=0.179. All P values were greater than 0.05, indicating that the distribution frequencies of the two loci in the control group were in accordance with the Hardy-Weinberg equilibrium, and the control group was representative of the population.

6. Association analysis of NF-κB pathway gene polymorphisms and susceptibility to HCV infection

Genotype distribution frequency of two SNP loci (rs1056890 and rs11820062) in NF-κB pathway genes and analysis of SNPs and HCV infection susceptibility and outcome by three genetic models (additive model, dominant model and recessive model) The relationship is shown in Table 4.

Table 4 Logistic regression analysis of SNPs in NF-κB signaling pathway genes and HCV infection susceptibility and outcome

Note: CI: confidence interval, HCV: hepatitis C virus, OR: odds ratio, SNP: single nucleotide polymorphism.

Group A: control group (uninfected group), Group B: self-limited clearance group, Group C: persistent infection group, Group (B+C): infected group (case group).

^a Logistic regression adjusted P value, OR value and 95% confidence interval, the adjustment factors are gender, age and infection route.

^b Logistic regression adjusted P value, OR value and 95% confidence interval, the adjustment factors are gender, age and infection route. The Bonferroni method was used to correct for multiple comparisons, and P<0.025 (0.05/2) was considered statistically different.

Data in bold indicates statistical difference.

After adjusting the confounding factors such as gender, age, ALT level and route of infection by Logistic regression, the results suggested that the risk of HCV infection in rs11820062AA genotype carriers increased, with a relative risk of 1.531 times, which was statistically significant (P=0.002). In the additive model, dominant model and recessive model, there is still a statistical correlation between the rs11820062A allele and HCV susceptibility, and the relative risk is 1.241 times (P=0.002), 1.354 times (P=0.003) and 1.346 times (P=0.018).

Taking gender, age, and route of infection as stratification factors, the rs11820062 locus in the NF-κB pathway gene and the susceptibility to HCV infection and the risk of outcome are further stratified, as shown in Table 5.

Table 5 Hierarchical analysis of the relationship between the rs11820062 site in the NF-κB signaling pathway gene and the susceptibility and outcome of HCV infection

Note: HCV: hepatitis C virus, CI: confidence interval, OR: odds ratio, SNP: single nucleotide polymorphism.

Group A: control group (uninfected group), Group B: self-limited clearance group, Group C: persistent infection group, Group (B+C): infected group (case group).

^a Logistic regression adjusted P value, OR value and 95% confidence interval, the adjustment factors are gender, age, infection route.

^b Logistic regression adjusted P value, OR value and 95% confidence interval, the adjustment factors are sex, age, infection route. Data in bold means statistically different.

According to the average age, the study population was divided into subgroups <50 years and ≥50 years for further analysis. As shown in Table 5, in the age <50 years old subgroup, female subgroup and paid blood donation subgroup, the rs11820062A allele was statistically associated with HCV susceptibility (all P<0.05), and the age <50 years old The relative risk of subgroup was 1.494 times (P=0.007), the relative risk of female subgroup was 1.353 times (P=0.020), and the relative risk of paid blood donation subgroup was 1.494 times (P=0.009).

7. Association analysis of NF-κB pathway gene polymorphisms and HCV self-limited clearance

After adjusting the confounding factors such as gender, age, ALT level, viral genotype and infection route by Logistic regression, no significant correlation was found between the genotype distribution frequency of rs1056890 and rs11820062 and the self-limited clearance of HCV infection (all P>0.05, See Table 4, Table 5).

8. Prediction of the secondary structure of the rs11820062 site

The wild-type and mutant mRNA sequences of the rs11820062 site of the RelA gene (50 bp upstream and downstream of the mutant position) are shown in SEQ ID NO.15 and SEQ ID NO.16, respectively.

In order to predict the impact of the mutation at rs11820062 on the RelA gene, we used the online tool RNAfoldWeb Server ( http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ) for the secondary analysis of its mRNA The structure was predicted, and the results are shown in Figure 2. The location of the mutation is indicated by the arrow in the figure. In Figure 2, the lowest energies of the secondary structures (structures with the minimum base pair distance) of the mRNA gravity centers of different allele types are: wild type: -22.50kcal/mol (Figure 2A) and -27.70kcal/mol ( Figure 2B). The results show that the energy required to maintain the structure is different before and after the mutation of this site, which may affect the binding of transcription factors near this site and the post-transcriptional regulation of RelA gene. It is speculated that it may be the potential link between rs11820062 site and HCV susceptibility molecular mechanism.

Therefore, the applicant believes that the single nucleotide polymorphism of RelA gene rs11820062 is related to the susceptibility to hepatitis C. Products for detection or screening of single nucleotide polymorphisms associated with hepatitis C, such as substances that will detect single nucleotide polymorphisms or genotypes of rs11820062 (Taqman PCR system as described in Table 2) and other substances (such as other substances that detect single nucleotide polymorphisms or genotypes associated with hepatitis C) or instruments are combined to prepare products for screening hepatitis C susceptible persons or to detect or screen hepatitis C-related Single nucleotide polymorphism products have broad application prospects.

sequence listing

<110> The First Affiliated Hospital of Nanjing Medical University

Nanjing Medical University

Drum Tower Hospital Affiliated to Nanjing University School of Medicine

<120> Application of RelA Gene rs11820062 SNP in Preparation and Detection of Hepatitis C Susceptibility Products

<141> 2017-10-27

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Claims (9)

1. The application of the single nucleotide polymorphism or genotype of RelA gene rs11820062 in the human genome in the preparation of products for detection or screening of hepatitis C susceptibility. 2. The application of the RelA gene rs11820062 single nucleotide polymorphism or genotype in the human genome in the preparation of products for detection or screening of hepatitis C-related single nucleotide polymorphisms. 3. The application of the substance for detecting the single nucleotide polymorphism or genotype of RelA gene rs11820062 in the human genome in the preparation of products for detecting or screening hepatitis C susceptibility. 4. The application of the substance for detecting the single nucleotide polymorphism or genotype of RelA gene rs11820062 in the human genome in the production of products for detecting or screening single nucleotide polymorphisms related to hepatitis C. 5. The application according to claim 3 or 4, characterized in that the substance for detecting the single nucleotide polymorphism or genotype of the RelA gene rs11820062 in the human genome comprises TaqMan PCR reagents. 6 . The application according to claim 5 , wherein the TaqMan PCR reagent includes PCR primers and TaqMan MGB probes for amplifying the genomic DNA fragment of the RelA gene rs11820062 in the human genome. 7. application according to claim 6, is characterized in that, described PCR primer sequence is shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; Described TaqMan MGB probe sequence is shown in SEQ ID NO.3 respectively and shown in SEQ ID NO.4, and the 5' ends of SEQ ID NO.3 and SEQ ID NO.4 are labeled with fluorescent reporter groups, and the 3' ends are labeled with non-fluorescent quenching groups and DNA minor groove binders MGB group. 8. A TaqMan PCR reagent containing PCR primers and TaqMan MGB probes for amplifying the genomic DNA fragment of the RelA gene rs11820062 in the human genome. 9. TaqMan PCR reagent according to claim 8, is characterized in that, described PCR primer sequence is shown in SEQID NO.1 and SEQ ID NO.2 respectively; Described TaqMan MGB probe sequence is shown in SEQ ID NO. 3 and SEQ ID NO.4, and the 5' ends of both SEQ ID NO.3 and SEQ ID NO.4 are labeled with fluorescent reporter groups, and the 3' ends are labeled with non-fluorescent quenching groups and DNA minor groove binders MGB group.