Application of miRNA combined marker to preparation of kit for diagnosing or detecting HBV+ and LC-primary HCC

A marker and primary technology, applied in the field of molecular biology and medical diagnosis, can solve the problems of lack of pertinence and complex pathogenesis of liver cancer, achieve excellent sensitivity and specificity, convincing detection results, and reduce errors. Effect

Active Publication Date: 2021-12-07
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the pathogenesis of primary liver cancer is more complex, and different types of patients may have different biomarkers. Existing panels are generally aimed at all types of liver cancer patients and lack specificity.

Method used

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  • Application of miRNA combined marker to preparation of kit for diagnosing or detecting HBV+ and LC-primary HCC
  • Application of miRNA combined marker to preparation of kit for diagnosing or detecting HBV+ and LC-primary HCC

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Subject and Sample Extraction

[0020] The samples were provided by Singapore General Hospital (approved by Singapore SingHealth CIRB B, approval number CIRB Ref: 2007 / 447 / B). Among them, there were 34 HCC patients with HBV+ / LC-, and 89 non-HCC patients with HBV+ / LC-.

Embodiment 2

[0021] Example 2 Extraction of exosomes

[0022] Collect 2ml of peripheral blood from the subject with a disposable syringe, quickly transfer it to an EDTA anticoagulant tube, and mix well. Separate the plasma within 2 hours of collection. The specific steps are: 1) transfer the whole blood to a 2ml centrifuge tube, and centrifuge at 500g for 10min at room temperature; 2) take the upper plasma, and centrifuge at 2000g for 10min at 4°C; 3) take the upper plasma, 4°C, Centrifuge at 16,000 g for 10 min; 4) The separated plasma is directly used for exosome extraction or frozen in a -80°C refrigerator.

[0023] Exosomes were extracted according to the instructions of the exosome extraction kit Exo-Quick exosome precipitation solution (EXOQ5SA-1, SBI, United States). 1) Add the plasma to a centrifuge tube, and add Exo-Quick at a ratio of 63 μl for every 250 μl of plasma; 2) Centrifuge at 1500 g at 4°C for 30 minutes, discard the supernatant; 3) Centrifuge at 1500 g at 4°C for 5 min...

Embodiment 3

[0024] Example 3 Extraction of exosome miRNA

[0025] miRNA was extracted according to the operating instructions of the miRNeasy Serum / PlasmaKit kit (217184, QIAGEN, Germany). The specific steps are: 1) Add 5 times the volume of QIAzol LysisReagent to the plasma separated in Example 2, vortex or pipette to mix; 2) Incubate at room temperature for 5 minutes; 3) Add an equal volume of chloroform and shake for 15 seconds; 4) Incubate at room temperature for 2-3min; 5) Centrifuge at 12000g for 15min at 4°C; 6) Transfer the upper layer to a new centrifuge tube, add 1.5 times the volume of absolute ethanol, and mix by pipetting to obtain the sample; 7) Take 700μl sample to RNeasy MinElute Spin column (placed in a 2ml collection tube), cover the lid, centrifuge at room temperature>8000g for 15s, discard; 8) repeat step 7 once; 9) add 700μl RWTbuf to the RNeasy MinElute spin column, centrifuge at room temperature>8000g for 15s, discard ;10) Add 500μl RPE buf to the RNeasy MinElute s...

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Abstract

The invention discloses an application of a miRNA combined marker to preparation of a kit for diagnosing or detecting HBV+ and LC-primary HCC. The miRNA combined marker is hsa-miR-146a-5p and hsa-miR-154-5p which are derived from a blood plasma exosome. According to the method, the LC-primary HCC of HBV+ is detected, the pertinence is higher, and the detection result is more convincing.

Description

technical field [0001] The invention relates to the fields of molecular biology and medical diagnosis, in particular to the application of a miRNA combined marker in the preparation of a kit for diagnosing or detecting HBV+ and LC-primary HCC. Background technique [0002] Primary liver cancer is one of the malignant tumors with the highest morbidity and mortality worldwide. Hepatocellular carcinoma (HCC) is the main form of primary liver cancer, which usually evolves from chronic liver diseases such as cirrhosis and HBV infection. The five-year survival rate of early HCC is 50%-70%, while that of patients with advanced liver cancer is only 10%-19%. Therefore, early diagnosis and screening is the key to the treatment of liver cancer. Due to the lack of early diagnostic methods to effectively distinguish between early liver cancer patients and chronic liver disease patients, the current diagnosis of HCC mainly relies on imaging, serum AFP and tissue biopsy, which has a certa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 陈健翔董衡乔逸婷常存杰李骞陈晓玉高浩晋孙孟情
Owner HANGZHOU NORMAL UNIVERSITY
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