Primers, probes and detection kit for detecting human HCC (Hepatocellular Carcinoma) marker

A technology of liver cancer cells and markers, which is applied in the field of molecular biology, can solve the problems of lack of mature quantitative standards for new markers and limited sensitivity of detection, and achieve the effect of simple detection method, strong specificity and high sensitivity

Active Publication Date: 2014-01-29
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the traditional detection methods of liver cancer serum markers, including ELISA detection technology, have great limitations due to the limited sensitivity of detection and the lack of mature quantitative standards for new markers.

Method used

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  • Primers, probes and detection kit for detecting human HCC (Hepatocellular Carcinoma) marker
  • Primers, probes and detection kit for detecting human HCC (Hepatocellular Carcinoma) marker
  • Primers, probes and detection kit for detecting human HCC (Hepatocellular Carcinoma) marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Establishment of multiple real-time fluorescent quantitative PCR detection method for human early liver cancer cell markers

[0042] According to the nucleotide sequences encoding glypican and alpha-fetoprotein published on GenBank (Accession No.: AAB58754; AAH35972), specific primers and probes for multiple real-time fluorescent quantitative PCR detection were designed, And carry out different fluorescein labeling. The specific primers and TaqMan probe sequences are shown in Table 1:

[0043] Table 1 Specific primers and TaqMan probes

[0044]

[0045]

[0046] Method for detecting human liver cancer cells based on multiple real-time fluorescent quantitative PCR technology:

[0047] 1. The object of detection is cDNA formed by reverse transcription of mRNA encoding liver cancer cell marker proteins (glypican and alpha-fetoprotein): sample processing and total RNA extraction are performed with TRIzol Reagent kit. use The reverse transcription kit performs reverse t...

Embodiment 2

[0055] Example 2 Detection of cell lines Hep G2, Huh7.5, Huh7, Huh28 and L02

[0056] 1. Five kinds of cells are extracted with TRIzol Reagent kit for RNA extraction.

[0057] 2. Adopt Reverse transcription kit, reverse transcription of five kinds of RNA respectively.

[0058] 3. Take 18μl of PCR reaction mixture into the PCR reaction tube, and then add 2μl of cDNA solution respectively. Add 2μl of positive control cDNA solution to the set positive control well, and add negative control cDNA solution or 2μl ddH to the negative control well 2 O.

[0059] 4. Centrifuge gently, mix the sample evenly and drop it to the bottom of the tube, ready for multiple fluorescent quantitative PCR reaction. After setting according to the program, put the PCR reaction tube into a fluorescent quantitative PCR instrument for detection.

[0060] 5. Analyze the specific S curve and the Ct value of the sample to be tested to determine the result.

[0061] PCR reaction mixture includes: 1×Taq enzyme buffer,...

Embodiment 3

[0069] Example 3 Detection of human liver paraffin-embedded tissue

[0070] 1. Use after treatment of human liver paraffin-embedded tissue RNeasy FFPE kit for RNA extraction.

[0071] 2. Adopt The reverse transcription kit performs reverse transcription of RNA.

[0072] 3. Take 18μl of PCR reaction mixture into the PCR reaction tube, and then add 2μl of cDNA solution respectively. Add 2μl of positive control cDNA solution to the set positive control well, and add negative control cDNA solution or ddH to the negative control well 2 O2μl.

[0073] 4. Centrifuge gently, mix the sample evenly and drop it to the bottom of the tube, ready for multiple fluorescent quantitative PCR reaction. After setting according to the program, put the PCR reaction tube into a fluorescent quantitative PCR instrument for detection.

[0074] 5. Analyze the specific S curve and the Ct value of the sample to be tested to determine the result.

[0075] Treatment method of human liver paraffin-embedded tissue: ...

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Abstract

The invention provides primers and probes for specifically detecting human HCC (Hepatocellular Carcinoma) marker, including a primer and a probe (Seq ID No.1-3) for detecting glypican cDNA (complementary deoxyribonucleic acid) and a primer and a probe (Seq ID No.4-6) used for detecting alpha fetoprotein cDNA. The invention also provides a detection kit containing the primers and the probes and used for detecting the marker of human HCC by multiplex real-time fluorescence quantification PCR (Polymerase Chain Reaction). According to the invention, by virtue of the multiplex real-time fluorescence quantification PCR, alpha fetoprotein messenger RNA (AFP mRNA) and glypican messenger RNA (GPC3 mRNA) in human HCC at the mRNA level can be detected; the detection method is simple, quick, high in sensitivity and strong in specificity; the whole detection reaction only lasts for 100min. An important technical support is provided for the specific detection of canceration of human HCC.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to primers and probes for specific detection of human liver cancer cell markers and a detection kit thereof. Background technique [0002] Primary liver cancer (HCC) is one of the most common malignant tumors in the world, and its mortality rate ranks third among various tumors. Because the early symptoms of liver cancer are not obvious, the patients are often in the middle and late stages when discovered, and the cure rate of liver cancer is not high, and the prognosis evaluation effect is not ideal. Therefore, early detection and determination of new liver cancer diagnostic targets has important clinical value. Alpha-fetoprotein is a serological indicator of primary liver cancer. However, it has been clinically found that the false positive and false negative rates of alpha-fetoprotein (AFP) in patients with liver cancer are relatively high, and the missed diagnosis rate of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/158
Inventor 方向东蔡侃李泽夏程华杜夏
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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