Hepatitis b/hepatic cirrhosis microRNA molecular marker combination and purpose thereof

A technology for molecular markers and hepatitis B, which is applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc. It can solve the problems that microRNA expression changes have not been studied in depth, and achieve easy mass screening and simple operation , highly specific effects

Pending Publication Date: 2017-01-18
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression changes of microRNAs in patients with liver cirrhosis have not been studied in depth.

Method used

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  • Hepatitis b/hepatic cirrhosis microRNA molecular marker combination and purpose thereof
  • Hepatitis b/hepatic cirrhosis microRNA molecular marker combination and purpose thereof
  • Hepatitis b/hepatic cirrhosis microRNA molecular marker combination and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Collection and preparation of plasma samples

[0038] During June 2012-March 2014, 150 plasma samples meeting the criteria of healthy controls, 150 plasma samples meeting the above definition of hepatitis B and 150 plasma samples meeting the above definition of liver cirrhosis were obtained from the Affiliated Hospital of Capital Medical University in advance. Collected from Beijing You'an Hospital.

[0039] Collect 10ml of peripheral venous blood and anticoagulate with EDTA. The plasma collection process is as follows: put the whole blood sample in a centrifuge at 4°C and centrifuge at 1,500-3,000g for 15 minutes. Carefully transfer the upper plasma layer to a 1.5 mL RNase-free sterile centrifuge tube with a 200 μL pipette. Label each sample. Be sure to store the plasma sample in an ultra-low temperature (-80°C) refrigerator within 4 hours.

Embodiment 2

[0040] Example 2: Extraction of total RNA in plasma

[0041] Use the RNA extraction kit (Beijing Kuangbo Biotechnology Co., Ltd.) to extract total RNA from the plasma, and add 1 μl (20 nM) of External Control-1 (Shanghai Biotechnology Co., Ltd. Synthesized by Engineering Bioengineering Technology Co., Ltd.) to monitor the extraction quality of RNA in plasma. The concentration of extracted total RNA was determined using Thermo NanoDrop 2000c.

Embodiment 3

[0042] Example 3: Three-step method for quantitative detection of miRNA in plasma

[0043] (1) Add polyA tail:

[0044] i. Prepare a polyA-tailed reaction solution in an RNase-free PCR tube (Axygen, 200 μl), with a volume of 20 μl. 1 μl (20nM) of External Control-2 (synthesized by Shanghai Sangon Biotechnology Co., Ltd.) with the sequence 5'-TGAGCAACGCGAACAA-3' (EC2) was added to each 20 μl system to monitor the quality of miRNA tailing and reverse transcription.

[0045]

[0046] (Note: The enzymes used in this experiment are all products of Beijing Kuangbo Biotechnology Co., Ltd.)

[0047] ii. Put the PCR tube containing the prepared reaction solution into a PCR machine (Thermo) and incubate at 37° C. for 1 hour. (2) RT-PCR to obtain cDNA single strand:

[0048] i. Add 0.5 μl (0.5ng / μl) of RT-Primer (synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) to the reaction solution obtained in step (1) with the sequence of 5'-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTT...

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Abstract

The invention provides a hepatitis b /hepatic cirrhosis microRNA molecular marker combination, which comprises more than one micro RNA nucleic acid molecules selected from mir.146a-5p, miR-22-3P and miR-381.3P, and a purpose of the marker combination for preparing hepatitis b/hepatic cirrhosis infection diagnosis and/or prognosis assessment kit. Due to a significant difference between content of a microRNA molecular marker of a hepatitis b/hepatic cirrhosis patient in plasma/serum and content in plasma/serum in health control, the hepatitis b/hepatic cirrhosis patient can be effectively distinguished from people of the health control; a chronic hepatitis b/hepatic cirrhosis disease state can be comprehensively analyzed, which can be used for guiding the patient treatment and usage of medication. The invention also provides a diagnostic kit for guiding the hepatitis b/hepatic cirrhosis infection diagnosis and/or prognosis assessment. By using the hepatitis b/hepatic cirrhosis microRNA molecular marker and the diagnostic kit, infection diagnosis and/or prognosis assessment of the hepatitis b/hepatic cirrhosis patient can be guided, and the combination has the characteristics of simple operation, safety and no wound, high specificity, high sensitivity and easy massive screening.

Description

technical field [0001] The present invention relates to the field of hepatitis B / cirrhosis diagnosis and medication, in particular to a group of hepatitis B / cirrhosis plasma microRNA molecular marker combinations, and the combination is used for infection diagnosis and treatment of hepatitis B / cirrhosis patients. The use of prognosis assessment is used for early diagnosis of patients, timely treatment, curative effect evaluation and medication guidance; and then early treatment of hepatitis and liver cirrhosis to improve the cure rate and drug treatment efficiency, improve public health, and reduce medical resources. waste. Background technique [0002] microRNA (miRNA) is a research hotspot in recent years. It is a single-stranded small molecule RNA widely present in eukaryotes. It has no coding function, but it can bind to the flanking region of the gene sequence to repress or inhibit the translation of target mRNA. , has a high degree of conservation, timing and tissue s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/68
Inventor 李宁张卫红张永宏靳文静魏颖颖
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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