Detecting method of nucleic acid mass spectrum for liver cancer susceptibility gene early screening

Abstract

The invention relates to the field of gene detection, in particular to a detecting method of a nucleic acid mass spectrum for liver cancer susceptibility gene early screening. The detecting method ischaracterized by screening out a combination of SNPs (Single Nucleotide Polymorphisms) of susceptibility gene related to liver cancer of Chinese people and gene mutation sites related to the liver cancer by considering the difference between liver cancer gene spectra of Chinese population and European and American populations, and widely (high-throughput detecting sites and high-throughput detecting samples) screening and checking genetic markers related to the liver cancer by utilizing a nucleic acid mass spectrometer. According to the detecting method disclosed by the invention, the detection success rate is high, the technology reproducibility is good, the cost performance ratio is high, polygenic detection of a single small sample can be realized, and maximum use of the small sample can be met. The detecting method is based on MassARRAY nucleic acid spectrometry; compared with Sanger sequencing, the detecting method has the technical advantages of high accuracy and high sensitivity, the detecting result is stable, and the detection positive rate is increased.

Description

technical field

[0001] The invention belongs to the field of gene detection, and in particular relates to a nucleic acid mass spectrometry detection method for early screening of liver cancer susceptibility genes. Background technique

[0002] Primary liver cancer (Hepatocellular Carcinoma, HCC) is one of the most common and harmful malignant tumors in my country and even in the world. The annual new cases in my country account for about 45% of the world, and it is the third most fatal malignant tumor in the world. Although the curative effect of liver cancer has been greatly improved with the emergence of various new technologies and new treatments and the improvement of diagnosis and treatment, the annual mortality rate of liver cancer in my country is still as high as 20.40 / 100,000, accounting for about 100% of the death rate of liver cancer in the world. 1 / 2.

[0003] The occurrence of liver cancer is a complex process involving multiple genes and factors, and is the resu...

Examples

Embodiment 1

[0140] Example 1: Feasibility analysis of SNPs of human liver cancer susceptibility-related genes and screening of gene mutation sites related to liver cancer

[0141] The inventors of the present invention searched NCBI domestic and foreign genome-wide association study (genome-wide association study, GWAS) research literature and related patents (patent literature 5 and non-patent literature 11, etc., including but not limited to This), while searching the GWAS Catalog database and cancer-related databases such as COSMIC, CIViC, ICGC, and MYCANCER GENOME, screened out more than 1,000 positive samples from the Asian population and more than 500 positive samples from the Chinese population in the GWAS study. The inventors screened and evaluated the liver cancer-related sites verified in the research, and selected 4 liver cancer hotspot mutations and 23 single nucleotide polymorphism sites that are significantly related to the risk of liver cancer in Asian populations , and are...

Embodiment 2

[0160] Embodiment 2: Detection of liver cancer cell lines by the technology of the present invention

[0161] 1. Research object

[0162] It is feasible to select 6 liver cancer cell lines (all of which can be purchased from ATCC) with known mutation sites SNU-475, C3A, PLC / PRF / 5, SK-HEP-1, SNU-387, and SUN-449 for the detection method For sex analysis, normal human genome DNA (gDNA) (extracted from the oral mucosa tissue of normal people in Wuhan General Hospital of Guangzhou Military Region) was set up as a negative control, and water (H 2 0) as a blank control.

[0163] 2. Experimental steps

[0164] (1) Extract DNA to obtain a DNA extract;

[0165] (2) As shown in Table 1, respectively mix 19 pairs of amplification primers of P1 group, 7 pairs of amplification primers of P2 group and 1 pair of amplification primers of P3 group, and add the mixed specific amplification primers to DNA Extract the solution, and then carry out PCR amplification reaction in the amplificatio...

Embodiment 3

[0190] Example 3: Detection of SNPs and mutation sites of genes related to early diagnosis of liver cancer in peripheral blood samples

[0191] 1. Research object

[0192] In this study, peripheral venous blood samples from Wuhan General Hospital of Guangzhou Military Command were used for genetic testing of samples, and the number of cases was 30. The control group was a sample of healthy people from local health checkups.

[0193] 2. Experimental steps

[0194] The specific steps of applying the technology of the present invention to detect the SNPs and mutation sites of genes related to the early diagnosis of liver cancer in peripheral blood samples are as follows:

[0195] (1) Use the rapid extraction kit to extract cfDNA:

[0196] (a) Take 200 μl of collected peripheral blood and put it into a 1.5ml centrifuge tube. Add 200 μl of binding solution CB, shake it upside down vigorously immediately, and mix well, then add 20ul proteinase K (20mg / ml) solution, shake it upsi...

technical field

[0001] The invention belongs to the field of gene detection, and in particular relates to a nucleic acid mass spectrometry detection method for early screening of liver cancer susceptibility genes. Background technique

[0002] Primary liver cancer (Hepatocellular Carcinoma, HCC) is one of the most common and harmful malignant tumors in my country and even in the world. The annual new cases in my country account for about 45% of the world, and it is the third most fatal malignant tumor in the world. Although the curative effect of liver cancer has been greatly improved with the emergence of various new technologies and new treatments and the improvement of diagnosis and treatment, the annual mortality rate of liver cancer in my country is still as high as 20.40 / 100,000, accounting for about 100% of the death rate of liver cancer in the world. 1 / 2.

[0003] The occurrence of liver cancer is a complex process involving multiple genes and factors, and is the resu...

Examples

Embodiment 1

[0140] Example 1: Feasibility analysis of SNPs of human liver cancer susceptibility-related genes and screening of gene mutation sites related to liver cancer

[0141] The inventors of the present invention searched NCBI domestic and foreign genome-wide association study (genome-wide association study, GWAS) research literature and related patents (patent literature 5 and non-patent literature 11, etc., including but not limited to This), while searching the GWAS Catalog database and cancer-related databases such as COSMIC, CIViC, ICGC, and MYCANCER GENOME, screened out more than 1,000 positive samples from the Asian population and more than 500 positive samples from the Chinese population in the GWAS study. The inventors screened and evaluated the liver cancer-related sites verified in the research, and selected 4 liver cancer hotspot mutations and 23 single nucleotide polymorphism sites that are significantly related to the risk of liver cancer in Asian populations , and are...

Embodiment 2

[0160] Embodiment 2: Detection of liver cancer cell lines by the technology of the present invention

[0161] 1. Research object

[0162] It is feasible to select 6 liver cancer cell lines (all of which can be purchased from ATCC) with known mutation sites SNU-475, C3A, PLC / PRF / 5, SK-HEP-1, SNU-387, and SUN-449 for the detection method For sex analysis, normal human genome DNA (gDNA) (extracted from the oral mucosa tissue of normal people in Wuhan General Hospital of Guangzhou Military Region) was set up as a negative control, and water (H 2 0) as a blank control.

[0163] 2. Experimental steps

[0164] (1) Extract DNA to obtain a DNA extract;

[0165] (2) As shown in Table 1, respectively mix 19 pairs of amplification primers of P1 group, 7 pairs of amplification primers of P2 group and 1 pair of amplification primers of P3 group, and add the mixed specific amplification primers to DNA Extract the solution, and then carry out PCR amplification reaction in the amplificatio...

Embodiment 3

[0190] Example 3: Detection of SNPs and mutation sites of genes related to early diagnosis of liver cancer in peripheral blood samples

[0191] 1. Research object

[0192] In this study, peripheral venous blood samples from Wuhan General Hospital of Guangzhou Military Command were used for genetic testing of samples, and the number of cases was 30. The control group was a sample of healthy people from local health checkups.

[0193] 2. Experimental steps

[0194] The specific steps of applying the technology of the present invention to detect the SNPs and mutation sites of genes related to the early diagnosis of liver cancer in peripheral blood samples are as follows:

[0195] (1) Use the rapid extraction kit to extract cfDNA:

[0196] (a) Take 200 μl of collected peripheral blood and put it into a 1.5ml centrifuge tube. Add 200 μl of binding solution CB, shake it upside down vigorously immediately, and mix well, then add 20ul proteinase K (20mg / ml) solution, shake it upsi...

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