CN106834455B - Kit and method for detecting liver cancer susceptibility gene of HBeAg negative HBV chronic infection cirrhosis patient - Google Patents
Kit and method for detecting liver cancer susceptibility gene of HBeAg negative HBV chronic infection cirrhosis patient Download PDFInfo
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Abstract
The invention provides a kit and a method for detecting whether a HBeAg negative HBV chronic infection cirrhosis patient is a liver cancer susceptible population, which relate to the field of biological detection and comprise an amplification reaction solution of a specific primer for amplifying DNA (deoxyribonucleic acid) containing SNP (Single nucleotide polymorphism) sites in a sample; a purification reaction solution comprising an SAP enzyme for purifying the DNA; when the detection result of the obtained rs187084 locus of the TLR9 gene shows CT or/and TT genotype, the extension reaction solution of the single-base extension primer is used for obtaining the base detection result of the rs187084 locus of the TLR9 gene, and a person suffering from chronic HBV infection cirrhosis with HBeAg negative is suggested to be a person susceptible to liver cancer. The specific primer and the extension primer provided by the invention can accurately amplify and extend a target gene, have high sensitivity and good specificity, can provide a basis for judging whether a HBeAg negative HBV chronic infection cirrhosis patient can develop hepatocellular carcinoma, are simple to operate and are beneficial to monitoring a prognosis target.
Description
Technical Field
The invention relates to the field of medical treatment and health, in particular to a kit and a method for detecting liver cancer susceptibility genes of a HBeAg negative HBV chronic infection cirrhosis patient.
Background
Currently, Hepatitis B Virus (HBV) infection is a worldwide public health problem. According to the statistics of the world health organization, 4 hundred million HBV infected people exist in the world, and Chinese HBV carriers account for 1/3 in the world. Among them, 10% of adults and about 80% -90% of children develop chronic hepatitis B after infection, while patients with chronic hepatitis B die of cirrhosis complicated by chronic hepatitis B and a large part of primary hepatocellular carcinoma, which is determined by the interaction of multiple factors and various factors, and the factors influencing the outcome of HBV infection include viral factors (such as viral genotype, viral load and viral variation); the immune status of the host (e.g., age at infection, presence or absence of other viral co-infection) and the genetic factors of the host. In particular, the polymorphism of host antiviral immunity-related genes largely determines the clinical outcome after HBV infection, but no research concerning the relationship between the gene polymorphism in TLR pathway and the disease progression of HBeAg-negative HBV chronically infected cirrhosis patients is available for suggesting the trend of disease progression of HBeAg-negative patients.
Tlr (toll like receptor) is a member of the Pattern Recognition Receptor (PRR) family, an ancient family of natural immune receptors with highly conserved sequences. It exists on the cell surface of human and is used as the main pattern recognition receptor and is involved in the innate immune response of the body. TLRs also play an important role in the human adaptive immune response (T, B cell response), which is a bridge connecting innate immunity and adaptive immunity. TLRs mediate specific signaling pathways, form a complex, associative network, specifically recognize a large number of pathogens and tissue metabolites, and activate innate and adaptive immunity through a series of signal transduction. TLRs are expressed in various liver tissue cells, after a human body is infected with hepatitis viruses, the TLRs activate intracellular signal transduction pathways by recognizing pathogen-related molecular modes such as virus double-stranded DNA, single-stranded RNA or non-methylated acid cytidine acyl, activate immune-related cells, generate proinflammatory factors and antiviral cytokines, and clear the viruses and simultaneously initiate liver cell inflammatory reaction. At present, 13 TLRs (TLR 1-TLR 13) have been identified in mammals, and 11 TLRs (TLR 1-TLR 11) exist in human bodies.
TLRs 3, 9 are both located on the intracellular endosome, lysosomal surface, and can recognize viral DNA. In HBV transgenic mice, poly i: both C-activated TLR3 and CpG-DNA containing ligand-activated TLR9 can cause IFN-mediated inhibition of HBV replication. The DNA chromosomes of the hepatitis B viruses A to H all contain CpG sequences recognized by TLR9, and HBV has an inhibitory effect on TLR 9-mediated immune response, which is also one of the mechanisms of chronic infection caused by escape of immune clearance of HBV. rs187084 is located in the promoter sequence of TLR9, and Hamann et al have shown that rs187084 site polymorphism may create a new binding site for transcription factors and may be functionally related to TLR 9. However, the relation between the rs187084 site of the TLR9 promoter sequence and the clinical outcome after HBV infection is not given, and the application of the rs187084 site to clinical prediction after HBV infection and the like is not given.
Disclosure of Invention
In order to find the kit in advance at the initial stage of infection, assist in treating patients and avoid accelerating the liver disease process from being converted into serious liver disease and even liver cancer, the invention provides a kit and a method for detecting liver cancer susceptibility genes of HBeAg negative HBV chronic infection cirrhosis patients, and the method mainly relies on rs187084 locus of TLR9 gene as a prompting standard to realize the trend prompt of the disease process. The method is simple to operate and is beneficial to monitoring the prognosis target.
In order to achieve the object of the present invention, the first aspect of the present invention provides a kit for detecting a liver cancer susceptibility gene of a person who has been chronically infected with HBeAg negative HBV and has cirrhosis, comprising:
an amplification reaction solution comprising a specific primer for amplifying a DNA containing an SNP site in a sample;
a purification reaction solution comprising an SAP enzyme for purifying the DNA;
and (3) extension reaction liquid of a single-base extension primer for obtaining a base detection result of the rs187084 locus of the TLR9 gene.
Wherein, when the detection result of the obtained rs187084 locus of the TLR9 gene shows CT or/and TT genotype, the HBeAg negative HBV chronic infection cirrhosis patients are suggested to be liver cancer susceptible people.
Wherein the liver cancer is hepatocellular carcinoma.
In particular, the detection of the rs187084 site of the TLR9 gene comprises:
amplifying a section of DNA containing SNP sites on a sample by using a specific primer and PCR amplification reaction liquid, and removing residual deoxyribonucleoside triphosphate and the primer in a PCR system by using SAP enzyme to obtain a sample DNA fragment;
adding a single-base extension primer, and carrying out single-base extension termination reaction on the sample DNA by adopting a single-base extension termination reaction solution to obtain a target gene for SNP analysis;
detecting the molecular weight difference between the extension product and the non-extension primer by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and determining the base of the rs187084 locus of the TLR9 gene;
and obtaining a judgment result of whether the HBeAg negative HBV chronic infection cirrhosis patient is a liver cancer susceptible patient or not according to the rs187084 locus base result of the TLR9 gene.
In particular, the specific primers comprise:
a forward primer: 5'-ACGTTGGATGTATTCCCCTGCTGGAATGTC-3', respectively;
reverse primer: 5'-ACGTTGGATGTTACTATGTGCTGGGCACTG-3' are provided.
Wherein the single base extension primer is: 5'-AAAAGATCACTGCCCT-3' are provided.
In particular, the reaction conditions for performing amplification using the amplification reaction solution are: pre-denaturation at 95 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 60s, 45 cycles, extension at 72 ℃ for 3min, and storage at 10 ℃.
Wherein, the PCR amplification reaction system further comprises: 10 buffer 0.625ul, Mg2+0.325ul, dNTP 0.25ul, Forward Primer 1ul, Reverse Primer 1ul, DNA 1ul (10ng-20ng), and Hotstar taq enzyme 0.2 ul.
In particular, the reaction conditions using the purified reaction solution are: storing at 37 deg.C for 40min, 85 deg.C for 5min, and 10 deg.C.
Wherein the SAP enzyme comprises: 1.53ul of triple distilled water, 0.17ul of Buffer and 0.3ul of SAP.
In particular, the reaction conditions using the single-base extension termination reaction solution are: 94 ℃ 30s, 94 ℃ 5s, 52 ℃ 5s, 80 ℃ 5s, 4 internal cycles (52 ℃ 5s, 80 ℃ 5s), 39 external cycles (94 ℃ 5s, 52 ℃ 5s, 80 ℃ 5s), 72 ℃ 3min, 10 ℃ storage. .
Wherein the single-base extension termination reaction solution comprises: triple distilled water 1.36, 10 × iplex buffer 0.2ul, Terminator mix 0.1ul, Primer 0.02, Thermo sequence 0.02 ul.
In order to achieve the technical object of the present invention, in a further aspect, the present invention provides a method for prompting whether a person with chronic cirrhosis due to HBeAg-negative HBV infection is a person with susceptibility to liver cancer, wherein the rs187084 locus of the TLR9 gene is judged, and when the rs187084 locus of the TLR9 gene shows a CT or/and TT genotype, the person with chronic cirrhosis due to HBeAg-negative HBV infection is a person with susceptibility to liver cancer, and the cirrhosis can progress to hepatocellular carcinoma.
Wherein the determination of the rs187084 locus of the TLR9 gene comprises the following steps:
amplifying a section of DNA containing SNP sites on a sample by using a specific primer and PCR amplification reaction liquid, and removing residual deoxyribonucleoside triphosphate and the primer in a PCR system by using SAP enzyme to obtain a sample DNA fragment;
adding a single-base extension primer, and carrying out single-base extension termination reaction on the sample DNA by adopting a single-base extension termination reaction solution to obtain a target gene for SNP analysis;
detecting the molecular weight difference between the extension product and the non-extension primer by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and determining the base of the rs187084 locus of the TLR9 gene;
and obtaining a disease progress trend judgment result of the HBeAg negative patient according to the rs187084 locus base result of the TLR9 gene.
Wherein, the specific primer comprises:
a forward primer: 5'-ACGTTGGATGTATTCCCCTGCTGGAATGTC-3', respectively;
reverse primer: 5'-ACGTTGGATGTTACTATGTGCTGGGCACTG-3' are provided.
Wherein the single base extension primer is: 5'-AAAAGATCACTGCCCT-3' are provided.
The invention has the beneficial effects that:
1. the specific primer and the extension primer provided by the invention can be used for accurately amplifying and extending a target gene, and have high sensitivity and good specificity.
2. The kit provided by the invention can accurately detect the genotype of the rs187084 locus of the TLR9 gene, and provides an accurate basis for judging whether the cirrhosis of a person with HBeAg negative HBV chronic infection cirrhosis can develop into hepatocellular carcinoma.
3. In order to find the HBeAg negative HBV chronic infected person in advance at the initial stage of infection, assist in treating the patient and avoid delay of treatment opportunity of the infected HBeAg negative HBV chronic infected person, the method provided by the invention can accurately predict that the liver cirrhosis of the HBeAg negative HBV chronic infected person can be developed into hepatocellular carcinoma, and the method mainly relies on rs187084 locus of TLR9 gene as a prompt standard to realize the prompt of disease progression trend, is simple to operate and is beneficial to monitoring of prognosis target.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be described in further detail below with reference to specific embodiments.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.
The main experimental instruments used in this example:
high speed centrifuges, Beckman Coulter ltd, usa;
SEQUENOM MASSARRAY matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry platform matrix-assisted laser desorption ionization time-of-flight mass spectrometry (manufactured by Sequenom, USA);
-20 ℃ and-80 ℃ low temperature refrigerator; an electronic scale; an ultra-clean bench; a horizontal centrifuge; an electrophoresis apparatus, an electrophoresis tank; an electric heating constant temperature water bath box.
EXAMPLE 1 kit
1. Amplification reaction solution
A method for amplifying DNA containing SNP sites in a sample, comprising: 10 buffer 0.625ul, Mg2+0.325ul, dNTP 0.25ul, Forward Primer lumen, Reverse Primer 1ul, DNA 1ul (10ng-20ng), Hotstar taq enzyme 0.2 ul.
Wherein the Forward Primer (i.e., Forward Primer) is:
5’-ACGTTGGATGTATTCCCCTGCTGGAATGTC-3’。
wherein, the Reverse Primer (namely Reverse Primer) is:
5’-ACGTTGGATGTTACTATGTGCTGGGCACTG-3’。
further, the reaction conditions using the amplification reaction solution were: 15min at 95 ℃; at 94 deg.C for 20s, at 56 deg.C for 30s, at 72 deg.C for 60s for 45 cycles, at 72 deg.C for 3min, and at 10 deg.C.
2. Purifying the reaction solution
The DNA fragment used for purifying the amplified DNA fragment mainly comprises SAP enzyme, and specifically comprises the following components: 1.53ul of triple distilled water, 0.17ul of Buffer and 0.3ul of SAP.
Further, the reaction conditions using the purified reaction solution were: storing at 37 deg.C for 40min, 85 deg.C for 5min, and 10 deg.C.
3. Extension reaction solution
The method is used for obtaining a base detection result of an rs187084 locus of a TLR9 gene, and specifically comprises the following steps: triple distilled water 1.36, 10 × iplex buffer 0.2ul, Terminator mix 0.1ul, Primer 0.02, Thermo sequence 0.02 ul.
Wherein the Primer (i.e. single base extension Primer) is: 5'-AAAAGATCACTGCCCT-3' are provided.
Further, the reaction conditions for carrying out the single base extension reaction using the extension reaction solution are: 94 ℃ 30s, 94 ℃ 5s, 52 ℃ 5s, 80 ℃ 5s, 4 internal cycles (52 ℃ 5s, 80 ℃ 5s), 39 external cycles (94 ℃ 5s, 52 ℃ 5s, 80 ℃ 5s), 72 ℃ 3min, 10 ℃ storage. .
Example 2 method for determining whether a HBeAg negative patient is a liver cancer-susceptible population
1. Specimen Collection and DNA extraction
A Blood clot (200. mu.l of the Blood clot dissolved in TES solution) was collected from the subject, and the whole genomic DNA was extracted using QIAGEN QIAampDNA Blood Mini Kit according to the protocol.
The specific operation steps are as follows:
1) first, 200. mu.l of AL buffer was added to a 1.5ml centrifuge tube. The patient's specimen was then gently pipetted and mixed, and 200. mu.l of the specimen was pipetted into a centrifuge tube containing lysis buffer (AL buffer).
2) Adding 20 mu l of QIAGEN protease or proteinase K into the centrifuge tube in the step 1), shaking and uniformly mixing for 15 seconds, centrifuging at 8000rpm/min for 10 seconds, and then putting into an electrothermal thermostatic waterbath box at 56 ℃ for water bath for 10 minutes.
3) And (4) centrifuging at a low speed to enable the liquid in the centrifuge tube cover to enter the centrifuge tube.
4) Adding 200 mul of absolute ethyl alcohol into the centrifuge tube, fully and uniformly mixing, shaking and uniformly mixing for 15 seconds, and then centrifuging again at 8000rpm/min for 10 seconds.
5) Transferring the material (i.e., the liquid including flocculent material) in the centrifuge tube of step 4) to a QIAamp spin column and placing it in a 2ml manifold tube with minimal adherence to the tube wall. The tube cover is tightly covered, and the tube is centrifuged at 8000rpm for 1 minute. The spin column is placed in another manifold and the used manifold can be discarded.
6) Open the column and add 500. mu.l of wash buffer (AW1 buffer) to the other manifold described in step 5) without wetting the walls of the tubes. The cover is closed, the mixture is centrifuged at 8000rpm for 1 minute, and the manifold is discarded. The spin column was placed in another clean manifold.
7) Open the column and add 500. mu.l of wash buffer (AW2 buffer) to another clean manifold as described in step 6). The cover is covered, and high-speed centrifugation is carried out for 3 minutes at 14000 r/min (if the maximum rotation speed of the centrifuge is only 13000, the high-speed centrifugation at 13000 r can be carried out for 5 minutes).
8) And (4) pouring the waste liquid in the collecting pipe in the step 7), continuously placing the centrifugal column on another collecting pipe, and emptying for 13000 revolutions or 14000 revolutions for 1 minute. The residual AW2 buffer was removed as much as possible.
9) The column was placed in an additional clean 1.5ml centrifuge tube and the manifold discarded. Carefully open the tube cap and carefully add 50-100. mu.l of a DNA-lysing solution (AE buffer). The mixture was left at room temperature for 10 minutes and then centrifuged at 8000rpm for 1 minute.
10) After DNA extraction, the sequence numbers are marked and stored in a refrigerator at-20 ℃ or-4 ℃ for later use.
2. Detection of site Gene polymorphism
Amplifying a section of DNA (about 50bp before and after the SNP locus) containing the SNP locus, removing residual deoxyribonucleoside triphosphate (dNTP) and a primer in a PCR system by SAP enzyme, adding a single-base extension primer, wherein the 3' terminal base of the primer is close to the SNP locus, and replacing the dNTP by four ddNTPs, so that the probe only extends one base at the SNP locus, and the connected ddNTPs correspond to alleles of the SNP locus. Detecting the molecular weight difference between the extension product and the non-extension primer by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to determine the basic group at the point, and the specific steps are as follows:
2.1PCR amplification
Carrying out PCR amplification reaction by using the specific primers and the obtained DNA sample, wherein the total reaction system is (namely an amplification reaction solution):
wherein the Forward Primer (i.e., Forward Primer) is:
5’-ACGTTGGATGTATTCCCCTGCTGGAATGTC-3’。
wherein, the Reverse Primer (namely Reverse Primer) is:
5’-ACGTTGGATGTTACTATGTGCTGGGCACTG-3’。
the amplification reaction system was set up according to the following procedure:
95℃15min；
94℃20s，
56℃30s，
60s 45 cycles at 72 ℃ for the reaction,
72℃3min，
storing at 10 deg.C.
Obtaining the target gene product after PCR amplification.
2.2 purification reaction
The SAP enzyme is used for removing the residual deoxyribonucleoside triphosphate (dNTP) and the primer in the PCR system, and the specific operation method is as follows:
placing the target gene product obtained in the step 3.1 after PCR amplification into a centrifuge for 3min at 3000 r/min, and preparing SAP enzyme Mix (namely a purification reaction solution) in a 1.5ml Tube according to the following sequence:
mix was evenly distributed over 12 well interconnected pipes. The rubber cover was gently uncovered, 2ul of Mix was added to each well with a 12-line gun and pumped back and forth to ensure mixing, the rubber cover was tightly covered and pressed tightly with round objects. Centrifuging at 3000 rpm for 3min, and covering with round object. SAP enzymatic digestion was performed in a PCR instrument according to the following procedure: storing at 37 deg.C for 40min, 85 deg.C for 5min, and 10 deg.C.
Obtaining the purified target gene.
2.3 Single base extension termination reactions
Putting the purified target gene into a centrifuge for 3min at 3000 r/m; a single-base extension reaction Mix (i.e., extension reaction solution) was prepared in a 1.5ml tube in the following order:
wherein the Primer (i.e. the extension Primer) is: 5'-AAAAGATCACTGCCCT-3' are provided.
Evenly distributing Mix in the 12-hole row pipes, and slightly uncovering the rubber cover; 2ul of Mix is taken by a 12-row gun and added into each hole, and is pumped back and forth to ensure uniform mixing, a rubber cover is tightly covered, and a round object is tightly pressed; centrifuging for 3min at 3000 rpm in a centrifuge, and covering the rubber cover tightly with round object; the single base extension reaction was performed in a PCR instrument according to the following procedure:
3 resin purification
Diluting the reaction product (9 u1 in total) obtained in step 3.3 by 3 times, and desalting with resin; and (3) putting the sample subjected to desalination treatment on a sample target, naturally crystallizing, and performing mass spectrum detection on a computer and collecting data.
4 data analysis
And when the detection result of the HBeAg negative HBV chronic infection cirrhosis patient shows that the rs187084 locus of the TLR9 gene is CT or TT genotype, judging that the patient is a liver cancer susceptible population.
Application examples
In this embodiment, the kit provided in embodiment 1 and the method provided in embodiment 2 are used to detect and analyze 686 samples, specifically as follows:
1 study object
All cases in this study met the diagnostic criteria of 2009 chronic hepatitis b guidelines, with the inclusion and exclusion criteria as follows:
1.1 Normal control group
The selection standard is Han nationality, and the age is more than or equal to 40 years old; HBsAg, anti-HBs, HBeAg and anti-HBe are all negative, and hepatitis B vaccine is not inoculated. No other hepatitis viruses; there is no systemic disease (such as kidney disease, heart disease, autoimmune disease, etc. have influence on the result judgment).
1.2HBV natural healing group
The selection standard is Han nationality, and the age is more than or equal to 40 years old; anti-HBs, anti-HBc positive or anti-HBs positive but not inoculated with hepatitis B vaccine; HBV DNA, anti-HAV, anti-HCV or anti-HDV, anti-HEV negative; without systemic diseases (such as kidney disease, heart disease, autoimmune disease, etc. having influence on the result judgment)
1.3HBV carriers
The selection standard is Han nationality, and the age is more than or equal to 40 years old; HBsAg is positive for at least half a year, and anti-HAV, anti-HCV or anti-HDV, anti-HEV are negative; ALT and AST detected normal ranges.
1.4 Chronic hepatitis B group
The selection standard is Han nationality, and the age is more than or equal to 40 years old; HBsAg is positive for at least half a year, and anti-HAV, anti-HCV or anti-HDV, anti-HEV are negative; ALT or AST is more than or equal to 60IU/L or 2 times of the upper limit of normal value or hepatosplenomegaly; or patients with chronic hepatitis confirmed by liver histopathology; clinically, the liver cirrhosis does not appear (such as portal hypertension, hyperfunction of spleen and the like); there is no systemic disease (such as kidney disease, heart disease, autoimmune disease, etc. have influence on the result judgment).
Since hepatitis B cirrhosis and hepatocellular carcinoma occur mostly after age 40, we selected the normal control group, the HBV natural recovery group, HBV carrier and chronic hepatitis B group all with age > 40 years.
1.5 group of liver cirrhosis
The selection standard is Han nationality, the age is not limited, and HBsAg is positive; anti-HAV, anti-HCV or anti-HDV, anti-HEV negative; clinically, the liver cirrhosis is manifested by the symptoms of cirrhosis (such as imaging diagnosis, varicose veins or bleeding of the esophagus and the stomach, ascites, edema of two lower limbs, hepatic encephalopathy, etc.); there is no systemic disease (such as kidney disease, heart disease, autoimmune disease, etc. which has influence on the result judgment).
1.6 hepatocellular carcinoma group
The selection standard is Han nationality, and the age is not limited; HBsAg positive; anti-HAV, anti-HCV or anti-HDV, anti-HEV negative; hepatocellular carcinoma was confirmed by liver histopathological examination or AFP and ultrasound, CT, or MRI; there is no systemic disease (such as kidney disease, heart disease, autoimmune disease, etc. which has influence on the result judgment).
The samples were subjected to HBV DNA quantification and HBsAg, Anti-HBs, HBeAg, Anti-HBe, Anti-HAV, Anti-HCV, Anti-HDV and Anti-HEV detection to exclude other types of hepatitis and to clarify the infection status of the patients, and the demographic and clinical characteristics as shown in Table 1 were obtained:
as can be seen from the table, the age of the normal control, the natural recovery group, the HBV carrier, the chronic hepatitis b group, the cirrhosis group, and the liver cancer group were 49.87 years old, 50.11 years old, 49.28 years old, 48.37 years old, 48.96 years old, and 49.31 years old, respectively (P ═ 0.425). The chronic hepatitis B group, the cirrhosis group and the hepatocellular carcinoma group had significantly more men than the healthy control group (72.4%, 76.8%, 82.7% vs 48.8%, P0.000). 67 cases of HBeAg positive patients and 64 cases of Anti-HBe positive patients in the chronic hepatitis B group; 45 cases of HBeAg positive patients and 48 cases of Anti-HBe positive patients in the liver cirrhosis group; 32 HBeAg positive patients and 66 Anti-HBe positive patients in the hepatocellular carcinoma group; 28 Anti-HBe positive patients in a natural healing group; HBeAg positive rates of the chronic hepatitis B group, the liver cirrhosis group and the liver cancer group are 46.2%, 40.5% and 29.4% respectively; the Anti-HBe positive rates in the natural recovery group, the carrier, the chronic hepatitis B group, the liver cirrhosis group and the hepatocellular carcinoma group were 19.3%, 69.8%, 44.1%, 43.2% and 60.6%, respectively. 94 cases of HBV DNA positive of chronic hepatitis B, 57 cases of HBV DNA positive of cirrhosis group and 80 cases of HBV DNA positive of hepatocellular carcinoma group have positive rates of 72.3%, 65.5% and 76.2% respectively.
2. 686 samples were detected and analyzed using the kit of example 1 and the method provided in example 2
2.1 different HBeAg states HBV infection outcome and TLRs pathway single site polymorphism relationship
In HBeAg (+) patients, the genotype distribution at each site was examined in the dominant, recessive, additive and multiplicative models, respectively. The results showed that the results in HBeAg (-) patients showed that the rs187084CT + TT genotype was significantly higher in the hepatocellular carcinoma group than in the cirrhosis group (χ 2 ═ 4.929, p ═ 0.026, OR ═ 2.468, 95% CI 1.071-5.683). Logistic regression results show that both the rs187084CT genotype and the TT genotype are independent risk factors for the development of cirrhosis to hepatocellular carcinoma in HBeAg (-) patients (p 0.043, OR 2.834, 95% CI 1.031-7.791; p 0.043, OR 3.000, 95% CI 1.034-8.702).
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> first Hospital of Beijing university
<120> kit and method for detecting whether HBeAg negative patient is liver cancer susceptible population
<160> 1
<170>PatentIn version 3.5
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ACGTTGGATGTATTCCCCTGCTGGAATGTC 30
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<213> Artificial sequence
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Claims (4)
1. The application of a reagent for detecting the TLR9 gene rs187084 locus in preparing a kit for detecting whether a HBeAg negative HBV chronic infection cirrhosis patient is a liver cancer susceptible population, which is characterized in that the kit comprises:
an amplification reaction solution comprising a specific primer for amplifying a DNA containing an SNP site in a sample;
a purification reaction solution comprising an SAP enzyme for purifying the DNA;
an extension reaction solution of a single base extension primer for obtaining a base detection result of the rs187084 locus of the TLR9 gene;
wherein, the specific primer comprises:
a forward primer: 5'-ACGTTGGATGTATTCCCCTGCTGGAATGTC-3', respectively;
reverse primer: 5'-ACGTTGGATGTTACTATGTGCTGGGCACTG-3', respectively;
wherein the single base extension primer is: 5'-AAAAGATCACTGCCCT-3' are provided.
2. The use of claim 1, wherein when the result of detecting the rs187084 locus of the TLR9 gene shows CT or/and TT genotype, the HBeAg negative HBV chronic infection cirrhosis patients are indicated to be liver cancer susceptible people.
3. The use of claim 2, wherein the detection of the rs187084 site of the TLR9 gene comprises:
amplifying a section of DNA containing SNP sites on a sample by using a specific primer and PCR amplification reaction liquid, and removing residual deoxyribonucleoside triphosphate and the primer in a PCR system by using SAP enzyme to obtain a sample DNA fragment;
adding a single-base extension primer, and carrying out single-base extension termination reaction on the sample DNA by adopting a single-base extension termination reaction solution to obtain a target gene for SNP analysis;
detecting the molecular weight difference between the extension product and the non-extension primer by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and determining the base of the rs187084 locus of the TLR9 gene;
and obtaining a judgment result of whether the HBeAg negative HBV chronic infection cirrhosis patient is a liver cancer susceptible patient or not according to the rs187084 locus base result of the TLR9 gene.
4. The use according to claim 1, wherein the reaction conditions for amplification using the amplification reaction solution are: pre-denaturation at 95 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 60s, 45 cycles, extension at 72 ℃ for 3min, and storage at 10 ℃.
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