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A human serum AFP-negative hepatocellular carcinoma detection kit

A specific, GAS5-AS1 technology, applied in the field of detection of human serum AFP-negative hepatocellular carcinoma, can solve the problems of missed detection, lack of effective detection methods for AFP-negative HCC patients, etc., and achieve the effect of improving survival rate

Active Publication Date: 2021-01-01
WUHAN UNIV
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] In view of the lack of effective detection methods for the diagnosis of AFP-negative HCC patients, and the increase of AFP in patients with chronic hepatitis B and liver cirrhosis, a certain proportion of AFP alone or combined with PIVAK-II detection will appear Therefore, it is necessary to find a more effective diagnostic index system to improve the detection rate of AFP-negative HCC

Method used

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  • A human serum AFP-negative hepatocellular carcinoma detection kit
  • A human serum AFP-negative hepatocellular carcinoma detection kit
  • A human serum AFP-negative hepatocellular carcinoma detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024]Plasma was collected from HCC (HCC) patients and healthy controls, including 59 cases of HCC (age: 55±9; sex: 55 males, 4 females), whose serum AFP was lower than 20ng / ml, and 72 healthy controls (age: 52 ±10; sex: 65 males, 7 females). Extract total RNA from plasma. The RNA extraction kit (RP4002) of Beijing Biotech Biotechnology Co., Ltd. was used, and the operation was performed according to the kit instructions. The brief description is as follows: ①Place EDTA anticoagulated whole blood in a low-temperature centrifuge, centrifuge at 2000×g for 5 minutes at 4°C. Separate plasma and blood cells. ② Carefully draw the upper layer of plasma and transfer it to a 1.5ml RNase-free EP tube. Note that this step should avoid suctioning the middle buffy coat, which contains white blood cells and platelets, which will cause contamination of RNA from sources outside the plasma. ③ Put the plasma in the RNase-free centrifuge tube back into the cryogenic centrifuge, and centrifug...

Embodiment 2

[0025] [Example 2] reverse transcription

[0026] Take 1 μg of total RNA for reverse transcription, using Takara (Dalian Bao Biotechnology Co., Ltd.) PrimeScript TM The RTreagent Kit with gDNA Eraser kit was carried out according to the steps recommended by the reagent manufacturer, as follows: ① Prepare the reaction reagents in an ice box, and the reaction system is shown in Table 1:

[0027] Table 1 Reverse transcription first step system

[0028]

[0029]

[0030] After mixing, centrifuge the reaction fraction to the bottom of the tube. ②After incubating at 42°C for 2 minutes, put the EP tube on ice again, and add the following reaction components:

[0031] Table 2 Reverse transcription second step system

[0032]

[0033] After mixing, the reaction components were spotted to the bottom of the tube. ③Place the reaction solution in a PCR instrument, first react at 37°C for 15 minutes, then react at 85°C for 5s. The reverse transcription experiment is completed....

Embodiment 3

[0034] [Example 3] Real-time fluorescent quantitative PCR

[0035] 1. The cDNA samples obtained in Example 3 were diluted 10 times to configure real-time fluorescent quantitative PCR systems. The system configuration is shown in Table 3:

[0036] Table 3 Fluorescent quantitative PCR reaction system

[0037]

[0038] 2. The detection target non-coding RNA and the corresponding internal reference primer sequences in the above table are shown in Table 4:

[0039] Table 4 forward primer and reverse primer feature table

[0040]

[0041] 3. Add the configured qRT-PCR solution into a real-time fluorescent quantitative PCR instrument (Bio-Red) for reaction. The reaction conditions for detecting target RNA in the above table are shown in Table 5:

[0042] Table 5 Fluorescent quantitative PCR conditions

[0043]

[0044]

[0045] 4, data statistics: the present invention carries out statistical analysis of data by IBMSPSS Statistics 21, evaluates the diagnostic value of...

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Abstract

The invention discloses a human serum AFP (Alpha Fetal Protein) negative liver cell cancer detection kit. A combined diagnosis kit is used for combined diagnosis on patients suffering from AFP negative hepatocellular carcinoma by detecting expression amounts of five RNAs (Ribonucleic Acids) of MiR-125b, MiR-205, SPRY4-IT1, GAS5-AS1 and circSMARCA5 in plasma by using a real-time quantitative PCR (Polymerase Chain Reaction) technique. The kit disclosed by the invention has the characteristics of high sensitivity, high specificity and high accuracy, patients suffering from AFP negative hepatocellular carcinoma can be effectively diagnosed, auxiliary diagnosis on early-stage hepatocellular carcinoma can be achieved, the survival rate of the patients can be increased, and the kit has remarkableclinical application values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for detecting AFP-negative hepatocellular carcinoma in human serum by combining five non-coding RNA markers miR-125b, miR-205, SPRY4-IT1, GAS5-AS1 and circSMARCA5. Background technique [0002] Liver cancer is one of the most common malignant tumors in my country, and its mortality rate ranks second among malignant tumors. 85%-90% of primary liver cancer is hepatocellular carcinoma (HCC). In China, the number of deaths due to HCC reaches 695,900 every year, which shows that HCC is one of the major diseases threatening human health. Liver cirrhosis is the most important risk factor for the formation of HCC, with an incidence rate of 80%-90%. , HCV) infection. The mortality rate of HCC has increased significantly in the past 20 years, and epidemiological studies have shown that the burden of drugs and economics brought by HCC will still increase significantly in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/166C12Q2600/178C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 涂建成陈珊珊景伟罗萍李南迪张献伟梁纯子邱石黎谭茜喻亚兰朱满王颖超刘雪芳周虎陈浩
Owner WUHAN UNIV
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