CN115961031B

CN115961031B - Nucleic acid combination and detection kit for liver cancer diagnosis or auxiliary diagnosis and application thereof

Info

Publication number: CN115961031B
Application number: CN202211011165.XA
Authority: CN
Inventors: 张良禄; 张玮; 董兰兰; 李国强; 昂学; 李婷婷
Original assignee: Wuhan Aimisen Life Technology Co ltd
Current assignee: Wuhan Amsen Life Science Technology Co Ltd
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2024-11-08
Anticipated expiration: 2041-06-02
Also published as: CN115961031A; CN113604563B; CN113604563A

Abstract

本发明公开了一种肝癌诊断或辅助诊断的核酸组合、检测试剂盒及其应用，涉及基因检测技术领域。以人GNB4基因的CpG岛为标志物，通过检测其甲基化的增加可以对肝癌进行诊断或辅助诊断，具有较高的灵敏度和特异性。采用本发明提供的检测试剂和试剂盒能有效地提高肝癌的检出率，从而满足肝细胞癌早筛早诊的临床需求。The present invention discloses a nucleic acid combination for liver cancer diagnosis or auxiliary diagnosis, a detection kit and its application, and relates to the field of gene detection technology. The CpG island of the human GNB4 gene is used as a marker, and liver cancer can be diagnosed or auxiliary diagnosed by detecting the increase of its methylation, with high sensitivity and specificity. The detection reagent and kit provided by the present invention can effectively improve the detection rate of liver cancer, thereby meeting the clinical needs of early screening and early diagnosis of hepatocellular carcinoma.

Description

Nucleic acid combination and detection kit for liver cancer diagnosis or auxiliary diagnosis and application thereof

Filing and applying for separate cases

The divisional application is based on China patent application with the application number 202110605465X, the application date 2021, 6 months and 2 days, and the invention name of 'a nucleic acid combination for diagnosing or assisting diagnosis of liver cancer, a detection kit and application thereof'.

Technical Field

The invention relates to the technical field of gene detection, in particular to a nucleic acid combination and a detection kit for liver cancer diagnosis or auxiliary diagnosis and application thereof.

Background

Primary liver cancer is the cause of death of the 4 th common malignant tumor and the 2 nd tumor in China. Primary liver cancer mainly comprises 3 different pathological types including hepatocellular carcinoma, intrahepatic cholangiocarcinoma and hepatocellular carcinoma-intrahepatic cholangiocarcinoma mixed type 3, wherein the hepatocellular carcinoma accounts for 85% -90%, and risk factors thereof include hepatitis b/c virus (HBV/HCV) infection, nonalcoholic fatty liver disease caused by obesity, chronic alcohol abuse and the like, which may directly lead to the development of liver cirrhosis, and the rate of liver cancer occurrence per year of liver cirrhosis patients is about 2% -4%.

The new occurrence rate of the primary liver cancer is 85.5 ten thousand worldwide every year, and 81 ten thousand cases of the primary liver cancer die worldwide every year. Survival rate of liver cancer patients in 5 years is 30.1% in japan, 27.2% in korea, and 17.4% in the united states. Prognosis of hepatocellular carcinoma is related to severity at the time of disease diagnosis. Because it was found that only 25% of primary liver cancer patients could undergo surgical resection at night. 85% of the patients with liver cancer in the first diagnosis are in the middle and late stages, and the early symptoms of the liver cell cancer are not obvious, so that the patients can reach the middle and late stages in diagnosis, and the surgical opportunity is lost. Early screening and early diagnosis are effective methods for reducing the morbidity and mortality of hepatocellular carcinoma, and are particularly important in a convenient, safe, rapid and high-throughput screening method.

The proposal recommended by the current diagnosis and treatment guidelines for the hepatocellular carcinoma in China is as follows: 1 liver ultrasonic examination and serum Alpha-fetoprotein (AFP) detection are carried out every 6 months on the high risk group, and liver cancer early screening is carried out. AFP is the only available blood marker for detecting and monitoring liver cancer at present, but the sensitivity and specificity of AFP are only 58.2% and 85.3%, and the clinical requirement of early screening and early diagnosis of hepatocellular carcinoma is difficult to meet. Imaging examinations remain quite difficult to confirm early hepatocellular carcinoma, and are susceptible to factors such as lesion size, machine sensitivity, and operator level in practical applications. In order to improve the early diagnosis proportion of people with high risk of hepatocellular carcinoma in China, a detection method with high sensitivity and specificity is urgently needed to popularize liver cancer early screening and improve the accuracy of early screening.

Currently, hepatocellular carcinoma lacks a detection means with high sensitivity and accuracy.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a nucleic acid combination for liver cancer diagnosis or auxiliary diagnosis, a detection kit and application thereof, so as to solve the technical problems.

DNA methylation is an early event in the occurrence of cancer, and the prior art shows that circulating tumor DNA (Ct DNA) in the blood has strong consistency with DNA in tumor tissue. Early noninvasive detection of liver cancer by detecting methylation of blood Ct DNA provides the possibility. In the process of canceration, the abnormality of DNA methylation level occurs in CpG island, the CpG island is mainly positioned in promoter and exon region of gene, and is a region rich in CpG dinucleotide, the length is 200-3000bp, and the content of G+C exceeds 50%.

The invention is realized in the following way:

The invention provides application of a reagent for diagnosing or assisting in diagnosing liver cancer in preparing a reagent kit for diagnosing or assisting in diagnosing liver cancer, wherein the reagent is used for detecting a molecular marker, the molecular marker is a CpG island of human GNB4 gene, and the CpG island is selected from the full length region or partial region of the following regions of the GNB4 gene:

Chr3:179450970-179451662bp plus strand and/or

Chr3 179451803-179451038bp negative strand.

The inventor uses methylation of CpG islands of human GNB4 genes as markers, diagnoses or assists diagnosis of liver cancer by detecting the increase of methylation levels of the CpG islands of the human GNB4 genes, has higher sensitivity and specificity, and provides a new idea and selection for diagnosis of liver cancer.

The detected region can be selected from the full-length region of the Chr3:179450970-179451662bp positive chain and the Chr3:179451803-179451038bp negative chain or a partial region in the region, and diagnosis or auxiliary diagnosis of liver cancer is realized through detection of the target region.

It should be noted that the positions of the sites or regions mentioned in the present invention are all reference genome of hg38 genome.

The base sequence of the forward strand of Chu 3:179450970-179451662bp is as follows (5 '-3'):

CACGCACGGGCTCGTGCTCTGAGTTCCTGGAAGGAGGCCTCGGGGAGTGACGAGAAACCAGGGGGGTCTGCAGGACTTGGACCGCCGACCGTTCCTCGCTCCCCGGGGCGAGCGGTCTGGACCGCCCGGGAAGTGCCTGCGCCGGCGGTCGTGGGGCCAGTTCCCGCGTGGCAGCTGGGCGCGACACAGGCGCGCCCTCCTCGTCCCTCCCGGGCAGCGTCGGCCGCCCGAGCCCGGGGAGACCCGCCCCGCCCCGCGCCGTCACCCGGGCCCCGTTCCGCAGGGGTGGCTCGCGGCGCCCCACGTCCCTGCGAGAAGCCCGGGATCGCTTCGCGGGGCGCACCGACGAGCCGCCGCTCGCGAGCTCGCCGCCTACCTGGAGGGAGCTCAGGCCCGCGTCGACCGCGCGCTGCCGGTGTCCGCTGGGCGCTCAGCAGCCCCTGGAGCGCGGAGCCGGCGTGGAGAGCGCAGCTCACAGCCGAGACCAGAGCCGCCGGCCACACCCAGTCCCGCACCTCCCAGCAGCCAACTCCGCGGCGCGCCGGAGCCGGGGCGGGGACGTGGCTGGAGGCGCGAGGCGCGAGGCACGAGGCGCGCGGGCCCGGCGGGGACGTGCCGGGGACGCGCAGACCCTCGGAGCGCGCGCAGCCCGGGCGGGGGGCGAAGGGAGCGGGCGCCGCGCGCAGCTTCTGC.

The base sequence on the 179451803-179451038bp negative strand of Chr3 is as follows (5 '-3'):

CCGGAAAGGAAACTGGCCACGCCACTTAGACGGGCGCTACTTAGCAGCGTGCCCCGGCGCCACACCAACAAGAAAACGAAACTGCCCGGGAATGAATGTTTTTCTTCTCCTTCTGGCAGGAAACGAGAGAGAAAATAGGAAGCAGAAGCTGCGCGCGGCGCCCGCTCCCTTCGCCCCCCGCCCGGGCTGCGCGCGCTCCGAGGGTCTGCGCGTCCCCGGCACGTCCCCGCCGGGCCCGCGCGCCTCGTGCCTCGCGCCTCGCGCCTCCAGCCACGTCCCCGCCCCGGCTCCGGCGCGCCGCGGAGTTGGCTGCTGGGAGGTGCGGGACTGGGTGTGGCCGGCGGCTCTGGTCTCGGCTGTGAGCTGCGCTCTCCACGCCGGCTCCGCGCTCCAGGGGCTGCTGAGCGCCCAGCGGACACCGGCAGCGCGCGGTCGACGCGGGCCTGAGCTCCCTCCAGGTAGGCGGCGAGCTCGCGAGCGGCGGCTCGTCGGTGCGCCCCGCGAAGCGATCCCGGGCTTCTCGCAGGGACGTGGGGCGCCGCGAGCCACCCCTGCGGAACGGGGCCCGGGTGACGGCGCGGGGCGGGGCGGGTCTCCCCGGGCTCGGGCGGCCGACGCTGCCCGGGAGGGACGAGGAGGGCGCGCCTGTGTCGCGCCCAGCTGCCACGCGGGAACTGGCCCCACGACCGCCGGCGCAGGCACTTCCCGGGCGGTCCAGACCGCTCGCCCCGGGGAGCGAGGAACGGTCGGCGGTCCAAGTCCTGCA.

In a preferred embodiment of the present invention, the CpG island is selected from the full-length region or a partial region of at least one of the following GNB4 genes: region 1, region 2, region 3, region 4, region 5, region 6, region 7, region 8 and region 9.

Wherein, region 1 is selected from the Chur 3:179450970-179451058bp positive strand, region 2 is selected from the Chur 3:179451090-179451203bp positive strand, region 3 is selected from the Chur 3:179451231-179451356bp positive strand, region 4 is selected from the Chur 3:179451379-179451472bp positive strand, region 5 is selected from the Chur 3:179451444-179451662bp positive strand, region 6 is selected from the Chur 3:179451803-179451709bp negative strand, region 7 is selected from the Chur 3:179451675-179451569bp negative strand, region 8 is selected from the Chur 3:179451457-179451278bp negative strand, region 9 is selected from the Chur 3:179451158-179451038bp negative strand;

In an alternative embodiment, the CpG island is selected from the full-length region or a partial region in at least one of the following regions of the GNB4 gene: region 2 positive strand, region 3 positive strand, region 8 negative strand and region 9 negative strand.

In practical embodiments, methylation detection may be performed in any one or any several of the above 9 regions, as desired. For example, regions 1-5 are selected for methylation detection.

Based on the present disclosure, one of ordinary skill in the art can employ any technique known in the art for detecting methylation of a combination of one or more of the above regions to diagnose liver cancer, regardless of the technique employed, and is within the scope of the present invention.

The invention also provides a nucleic acid combination for detecting the methylation level of a CpG island of the human GNB4 gene, wherein the CpG island is selected from the following full length region or partial region of the GNB4 gene:

Chr3:179450970-179451662bp plus strand and/or

The negative strand of 179451803-179451038bp is Chr 3;

preferably, the CpG island is selected from the full-length region or a partial region in at least one of the following regions of the GNB4 gene: region 1, region 2, region 3, region 4, region 5, region 6, region 7, region 8 and region 9;

Preferably, the CpG island is selected from the full-length region or a partial region in at least one of the following regions of the GNB4 gene: region 2 positive strand, region 3 positive strand, region 8 negative strand and region 9 negative strand. In the plasma sample, the methylation of the 4 areas is detected with the detection sensitivity of more than 85 percent on liver cancer and the detection specificity of more than 95 percent on liver cancer. In liver tissue samples, the methylation of the 4 areas is detected with the detection sensitivity of more than 95% on liver cancer and the detection specificity of 100% on liver cancer.

In a preferred embodiment of the present invention, the above nucleic acid combination for detecting the methylation level of CpG islands of the human GNB4 gene is at least one selected from the following nucleic acid combinations: nucleic acid combination 1 for detection region 1, nucleic acid combination 2 for detection region 2, nucleic acid combination 3 for detection region 3, nucleic acid combination 4 for detection region 4, nucleic acid combination 5 for detection region 5, nucleic acid combination 6 for detection region 6, nucleic acid combination 7 for detection region 7, nucleic acid combination 8 for detection region 8, nucleic acid combination 9 for detection region 9.

The base sequence of the nucleic acid combination 1 is shown as SEQ ID NO.1-3, the base sequence of the nucleic acid combination 2 is shown as SEQ ID NO.4-6, the base sequence of the nucleic acid combination 3 is shown as SEQ ID NO.7-9, the base sequence of the nucleic acid combination 4 is shown as SEQ ID NO.10-12, the base sequence of the nucleic acid combination 5 is shown as SEQ ID NO.13-15, the base sequence of the nucleic acid combination 6 is shown as SEQ ID NO.16-18, the base sequence of the nucleic acid combination 7 is shown as SEQ ID NO.19-21, the base sequence of the nucleic acid combination 8 is shown as SEQ ID NO.22-24, and the base sequence of the nucleic acid combination 9 is shown as SEQ ID NO. 25-27.

The above nucleic acid combinations are only one or several of the nucleic acid combinations provided by the inventors, and furthermore, in other embodiments, it is within the scope of the present invention to delete or add bases according to the above nucleic acid combinations. For example, the nucleic acid combinations 1 to 9 have a sequence identity of 80% or more with any one of the above nucleic acid combinations.

In a preferred embodiment of the application of the present invention, the nucleic acid composition includes a probe sequence, wherein the 5 'end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end of the probe sequence is labeled with a fluorescent quenching group.

In a preferred embodiment of the present invention, the fluorescent reporter group is HEX, FAM, TET, CF, JOE, TAMRA, ROX, CY, CY5, texas Red, NED, alexa inur or VIC, and the quencher group is MGB, TAMRA, BHQ1, BHQ2, BHQ3 or QSY. In other embodiments, the probe-labeled fluorescent reporter and quencher groups may also be adaptively adjusted as desired, and are not limited to the types defined above.

The invention also provides a detection reagent for diagnosing or assisting in diagnosing liver cancer, which comprises the nucleic acid combination for detecting the CpG island methylation level of the human GNB4 gene.

In a preferred embodiment of the application of the present invention, the detection reagent is a reagent for detecting the methylation level of CpG islands of the human GNB4 gene, and the means for detecting the methylation level comprises at least one of the following methods: methylation-specific PCR, bisulfite, methylation-specific microarray, whole genome methylation, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and flap endonuclease.

The invention also provides a detection kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleic acid combination for detecting CpG islands of human GNB4 genes or a detection reagent for diagnosing or assisting in diagnosing liver cancer.

In a preferred embodiment of the present invention, the test sample of the kit is a blood sample or a tissue sample taken from a subject to be tested.

In a preferred embodiment of the invention, the kit further comprises a buffer solution, dNTPs, a DNA polymerase and water.

The invention has the following beneficial effects:

the nucleic acid composition, the detection reagent and the kit provided by the invention take methylation of CpG islands of human GNB4 genes as markers, and can diagnose or assist diagnosis of liver cancer by detecting the increase of methylation levels, and have higher sensitivity and specificity. The detection reagent and the kit provided by the invention can effectively improve the detection rate of liver cancer, thereby meeting the clinical requirements of early screening and early diagnosis of hepatocellular carcinoma.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 1, wherein the nucleotide combination 1 comprises nucleotides shown in SEQ ID NO.1-3, and a specific sequence table 1. The nucleotide combination 1 can detect methylation of a forward strand (region 1) of the 179450970-179451058 region of the Chr3:3835 gene;

The base sequence of the plus strand of region 1 is as follows (5 '-3'):

CACGCACGGGCTCGTGCTCTGAGTTCCTGGAAGGAGGCCTCGGGGAGTGACGAGAAACCAGGGGGGTCTGCAGGACTTGGACCGCCGAC。

Example 2

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 2, wherein the nucleotide combination 2 comprises nucleotides shown in SEQ ID NO.3-6, and a specific sequence table 1. The nucleotide combination 2 can detect methylation of a forward strand (region 2) of the 179451090-179451203 region of the Chr3:3835 gene;

the base sequence of the positive strand of region 2 is as follows (5 '-3'):

ACCGCCCGGGAAGTGCCTGCGCCGGCGGTCGTGGGGCCAGTTCCCGCGTGGCAGCTGGGCGCGACACAGGCGCGCCCTCCTCGTCCCTCCCGGGCAGCGTCGGCCGCCCGAGCC.

example 3

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 3, wherein the nucleotide combination 3 comprises nucleotides shown in SEQ ID NO.7-9, and a specific sequence table 1. The nucleotide combination 3 can detect methylation of a forward strand (region 3) of the 179451231-179451356 region of the Chr3:3835 gene;

the base sequence of the forward strand of region 3 is as follows (5 '-3'):

TCACCCGGGCCCCGTTCCGCAGGGGTGGCTCGCGGCGCCCCACGTCCCTGCGAGAAGCCCGGGATCGCTTCGCGGGGCGCACCGACGAGCCGCCGCTCGCGAGCTCGCCGCCTACCTGGAGGGAGC.

Example 4

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 4, wherein the nucleotide combination 4 comprises nucleotides shown in SEQ ID NO.10-12, and a specific sequence table 1. The nucleotide combination 4 can detect methylation of a forward strand (region 4) of the 179451379-179451472 region of the Chr3:3835 gene;

The base sequence of the plus strand of region 4 is as follows (5 '-3'):

CTGCCGGTGTCCGCTGGGCGCTCAGCAGCCCCTGGAGCGCGGAGCCGGCGTGGAGAGCGCAGCTCACAGCCGAGACCAGAGCCGCCGGCCACAC。

Example 5

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 5, wherein the nucleotide combination 5 comprises nucleotides shown in SEQ ID NO.13-15, and a specific sequence table 1. The nucleotide combination 5 can detect methylation of the positive strand (region 5) of the 179451444-179451662 region of Chr3:3835 on the GNB4 gene;

the base sequence of the positive strand in region 5 is as follows (5 '-3'):

ACAGCCGAGACCAGAGCCGCCGGCCACACCCAGTCCCGCACCTCCCAGCAGCCAACTCCGCGGCGCGCCGGAGCCGGGGCGGGGACGTGGCTGGAGGCGCGAGGCGCGAGGCACGAGGCGCGCGGGCCCGGCGGGGACGTGCCGGGGACGCGCAGACCCTCGGAGCGCGCGCAGCCCGGGCGGGGGGCGAAGGGAGCGGGCGCCGCGCGCAGCTTCTGC.

example 6

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 6, wherein the nucleotide combination 6 comprises nucleotides shown in SEQ ID NO.16-18, and a specific sequence table 1. The nucleotide combination 6 can detect methylation of a negative strand (region 6) in the 179451803-179451709 region of the Chr3:3835 gene;

the negative strand base sequence of region 6 is as follows (5 '-3'):

CCGGAAAGGAAACTGGCCACGCCACTTAGACGGGCGCTACTTAGCAGCGTGCCCCGGCGCCACACCAACAAGAAAACGAAACTGCCCGGGAATGA。

Example 7

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 7, wherein the nucleotide combination 7 comprises nucleotides shown in SEQ ID NO.19-21, and a specific sequence table 1. The nucleotide combination 7 can detect methylation of the negative strand (region 7) of the 179451675-179451569 region of Chr3:3835 on the GNB4 gene;

The negative strand base sequence of region 7 is as follows (5 '-3'):

GAGAAAATAGGAAGCAGAAGCTGCGCGCGGCGCCCGCTCCCTTCGCCCCCCGCCCGGGCTGCGCGCGCTCCGAGGGTCTGCGCGTCCCCGGCACGTCCCCGCCGGGC.

Example 8

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 8, wherein the nucleotide combination 8 comprises nucleotides shown in SEQ ID NO.22-24, and a specific sequence table 1. The nucleotide combination 8 can detect methylation of a negative strand (region 8) of the 179451457-179451278 region of the Chr3:3835 gene;

the negative strand base sequence of region 8 is as follows (5 '-3'):

CTGGTCTCGGCTGTGAGCTGCGCTCTCCACGCCGGCTCCGCGCTCCAGGGGCTGCTGAGCGCCCAGCGGACACCGGCAGCGCGCGGTCGACGCGGGCCTGAGCTCCCTCCAGGTAGGCGGCGAGCTCGCGAGCGGCGGCTCGTCGGTGCGCCCCGCGAAGCGATCCCGGGCTTCTCGCAG.

Example 9

The embodiment provides a kit for diagnosing or assisting in diagnosing liver cancer, which comprises a nucleotide combination 9, wherein the nucleotide combination 9 comprises nucleotides shown in SEQ ID NO.25-27, and a specific sequence table 1. The nucleotide combination 9 can detect methylation of the negative strand (region 9) of the 179451158-179451038 region of Chr3:3835 on the GNB4 gene;

the negative strand base sequence of region 9 is as follows (5 '-3'):

CTGTGTCGCGCCCAGCTGCCACGCGGGAACTGGCCCCACGACCGCCGGCGCAGGCACTTCCCGGGCGGTCCAGACCGCTCGCCCCGGGGAGCGAGGAACGGTCGGCGGTCCAAGTCCTGCA

table 1 is a sequence listing of combinations of nucleic acids.

Example 10

This example provides a nucleic acid combination comprising the nucleotide combination 2 of example 2 and the nucleotide combination 3 of example 3.

Example 11

This example provides a nucleic acid combination comprising the nucleotide combination 8 of example 8 and the nucleotide combination 9 of example 9.

Example 12

This example provides a nucleic acid combination comprising the nucleotide combination 2 of example 2, the nucleotide combination 3 of example 3, the nucleotide combination 8 of example 8 and the nucleotide combination 9 of example 9.

Example 13

The present embodiment provides a method for diagnosing liver cancer using any one of the kits of embodiments 1 to 9, comprising the steps of:

(1) Plasma DNA extraction and transformation.

Firstly, 5mL of blood is centrifuged for 12 minutes at 1300 Xg to separate plasma, the plasma is stored in a refrigerator at-80 ℃ for standby, DNA in the plasma is extracted by using a magnetic bead method serum/plasma free DNA extraction kit (DP 709) of Tiangen biochemical technology (Beijing) limited company, and the extracted DNA is subjected to bisulphite conversion by EpiTech Bisulfite Kit, and the specific operation is described in a kit instruction. Upon conversion, unmethylated cytosine (C) is converted to uracil (U), the methylated cytosine is unchanged, uracil is paired with adenine (a) and cytosine is paired with guanine (G) in a subsequent PCR step, thereby effecting differentiation of the methylated from unmethylated sequences.

(2) Positive control, negative control preparation.

The positive control and the negative control of each gene are artificial synthetic sequences constructed on a carrier, the base composition of the artificial synthetic sequences is designed by referring to the sequence of a target fragment to be amplified, the positions of all cytosine C in the negative control are designed to be T, the C at the position of a CG dinucleotide in the positive control is designed to be T except the C at the position of the CG dinucleotide, and the nucleotides at other positions are identical with the target fragment to be amplified.

(3) PCR reaction

The PCR reaction system using beta-actin as an internal reference gene is shown in Table 2. Beta-actin is used as an internal reference gene, wherein the upstream primer of the beta-actin is as follows: AAGGTGGTTGGGTGGTTGTTTTG (SEQ ID NO. 28); the beta-actin downstream primer is: AATAACACCCCCACCCTGC (SEQ ID NO. 29); the beta-actin probe is as follows: GGAGTGGTTTTTGGGTTTG (SEQ ID NO. 30).

In this embodiment, the reporter group at the 5 'end of the probe for detecting the target region is FAM, the 3' end quenching group is MGB, the reporter group at the 5 'end of the β -actin probe is VIC, and the 3' end quenching group is BHQ1.

Table 2 PCR system.

As shown in Table 2, in detecting the methylation state of any of GNB4 region 1 to region 9 in a sample, only the primer probe corresponding to a certain region, the beta-actin primer probe, the buffer, dNTPs, DNase, sample DNA, and the like are added to the reaction system in the volumes shown in the table.

The PCR reaction conditions are shown in Table 3 below.

Table 3 PCR reaction conditions.

Ct value reading: after the PCR is completed, a baseline is adjusted, a fluorescence value before a minimum Ct value of a sample in one PCR is advanced by 1-2 cycles is set as a baseline value, and a threshold value is set at the inflection point of an S-type amplification curve to obtain Ct values of all genes of the sample.

And (3) quality control: and (3) synchronously detecting a negative control and a positive control in each detection, wherein the negative control is purified water, the positive control is an artificial plasmid containing beta-actin gene and target gene sequences, the concentration is 10 ³ copies/microliter, the negative control needs no amplification, the positive control has obvious index increase period, and the Ct value of the positive control is between 26 and 30. After the negative control, the positive control and the reference gene meet the requirements, the experiment is effective, and the next sample result can be judged. Otherwise, when the experiment is invalid, the detection is needed again.

Result analysis and interpretation method: under the same experimental conditions, when methylation of the same region is detected for different samples, if the Ct value of the sample is smaller, the methylation level of the detection region in the sample is higher. If the Ct value of a certain detection area on the sample is less than or equal to 38, the sample is considered to be methylation positive in the detection area, and if the Ct value of a certain detection area on the sample is more than 38, the sample is considered to be methylation negative in the detection area. Comparing the methylation detection result of the sample with the pathological result, and calculating the sensitivity and specificity of the methylation detection. Sensitivity is the proportion of methylation positives in samples with positive pathological results, and specificity is the proportion of methylation negatives in samples with negative pathological results.

Experimental example 1

A total of 143 liver tissue samples and 50 paracancerous samples were collected from the southern Hospital of the university of Wuhan, genomic extraction, bisulfite conversion were performed as described in the examples, and the methylation status of regions 1 to 9 was detected using the converted DNA as a template, respectively. As shown in table 4 below:

Table 4 detection sensitivity and specificity in liver tissue samples for regions 1-9.

As shown in the results of Table 4, in the tissue samples, the detection sensitivity of the regions 1-9 to the liver cancer sample is above 85%, wherein the detection sensitivity of the regions 2,3, 8 and 9 is optimal and is more than 95%; the detection specificity of the areas 1-9 to the tissues beside the liver cancer is more than 95%, wherein the detection specificity of the areas 2,3, 8 and 9 is 100%.

Experimental example 2

Blood samples of 120 healthy persons and blood samples of 95 liver cancer patients were collected from the southern hospitals of the university of martial arts in total, plasma separation, genome extraction, bisulfite conversion were performed according to the methods described in examples, and methylation states of regions 1 to 9 were detected using the converted DNA as a template, respectively. The sensitivity and specificity of the cancer sample and the normal sample were calculated using Ct value=38 as a critical value, as shown in table 5 below:

Table 5 detection sensitivity and specificity in plasma samples for regions 1-9.

From the results in table 5, it can be seen that, in the plasma samples, the detection sensitivities of the regions 1 to 9 to the liver cancer sample are all above 80%, wherein the detection sensitivities of the regions 2,3, 8 and 9 are all optimal and are all above 85%, and the detection sensitivities of the regions 3, 8 and 9 are all above 90%; the detection specificity of the areas 1-9 to the tissues beside the liver cancer is more than 95%, wherein the detection specificity of the areas 2,3, 8 and 9 is more than 98%.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a reagent for liver cancer diagnosis or auxiliary diagnosis in the preparation of a liver cancer diagnosis or auxiliary diagnosis kit, characterized in that the reagent is used for detecting the methylation level of a molecular marker selected from the full-length region of the GNB4 gene in the following regions: region 3 or region 8;

The GRCh38.p14 genome is taken as a reference genome, and the region 3 is selected from the Ch3: 179451231-179451356bp positive strand; said region 8 is selected from the group consisting of the Chr3:179451457-179451278bp negative strand; the reagent is selected from the following nucleic acid combinations: a nucleic acid combination 3 for detecting the region 3 or a nucleic acid combination 8 for detecting the region 8;

the nucleic acid combination 3 consists of a primer with a nucleotide sequence shown as SEQ ID NO.7-8 and a probe with a nucleotide sequence shown as SEQ ID NO.9, and the nucleic acid combination 8 consists of a primer with a nucleotide sequence shown as SEQ ID NO.22-23 and a probe with a nucleotide sequence shown as SEQ ID NO. 24.

2. A nucleic acid combination for detecting the methylation level of a human GNB4 gene, wherein the nucleic acid combination detects a full-length region selected from the group consisting of: region 3 or region 8;

said region 3 is selected from the group consisting of the Chr3:179451231-179451356bp plus strand; said region 8 is selected from the group consisting of the Chr3:179451457-179451278bp negative strand; the nucleic acid combination is selected from the following nucleic acid combinations: a nucleic acid combination 3 for detecting the region 3 or a nucleic acid combination 8 for detecting the region 8;

The nucleotide sequence of the nucleic acid combination 3 is shown as SEQ ID NO. 7-9, and the nucleotide sequence of the nucleic acid combination 8 is shown as SEQ ID NO. 22-24.

3. A diagnostic or diagnostic-assisted detection reagent for liver cancer, comprising the nucleic acid combination for detecting the methylation level of human GNB4 gene according to claim 2.

4. The reagent for detecting a liver cancer diagnosis or auxiliary diagnosis according to claim 3, wherein the reagent for detecting a methylation state of human GNB4 gene comprises at least one of the following means: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray or methylation-specific high resolution dissolution profile.

5. A kit for diagnosing or aiding diagnosis of liver cancer, comprising the nucleic acid combination for detecting the methylation level of the GNB4 gene of human according to claim 2 or the diagnostic reagent for diagnosing or aiding diagnosis of liver cancer according to any one of claims 3 to 4.

6. The kit for diagnosis or auxiliary diagnosis of liver cancer according to claim 5, wherein the test sample of the kit is a blood sample or a tissue sample taken from a subject to be tested.

7. The kit for diagnosis or auxiliary diagnosis of liver cancer according to claim 5 or 6, wherein the kit further comprises a buffer, dNTPs, DNA polymerase and water.