Hepatitis B microRNA molecular marker composition and application thereof

A molecular marker, hepatitis B technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial determination / inspection, etc., to achieve the effect of easy mass screening, high specificity, and simple operation

Inactive Publication Date: 2014-12-24
北京旷博生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no research on the expression changes of miRNA before and after treatment of hepatitis B patients and whether the expression changes are related to the treatment and prognosis of hepatitis B. In clinical research, it is necessary to find out the effective way to judge the efficacy and guidance of hepatitis B after treatment. miRNA markers of clinical drug withdrawal

Method used

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  • Hepatitis B microRNA molecular marker composition and application thereof
  • Hepatitis B microRNA molecular marker composition and application thereof
  • Hepatitis B microRNA molecular marker composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Collection and preparation of plasma samples

[0036] Between June 2012 and March 2014, 150 plasma samples meeting the criteria of healthy controls and 150 plasma samples meeting the above definition of hepatitis B were pre-collected from Beijing You'an Hospital affiliated to Capital Medical University.

[0037] 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

[0038] Example 2: Extraction of total RNA in plasma

[0039] Use the RNA extraction kit (Beijing Kuangbo Biotechnology Co., Ltd.) to extract total RNA from the plasma, and add 1 μl (20 nM) of the sequence 5'-CAACCTCCTAGAAAGAGTA-3' (SEQ ID NO: 37) to each 250 μl of plasma. 1 (synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) to monitor the extraction quality of RNA in plasma. The concentration of extracted total RNA was measured using Thermo NanoDrop2000c.

Embodiment 3

[0040] Example 3: Three-step method for quantitative detection of miRNA in plasma

[0041] (1) Add polyA tail:

[0042] i. Prepare a polyA-tailed reaction solution in an RNase-free PCR tube (Axygen, 200 μl), with a volume of 20 μl. Add 1 μl (20nM) of External Control-2 (synthesized by Shanghai Sangon Biotechnology Co., Ltd.) with the sequence 5'-TGAGCAACGCGAACAA-3' (SEQ ID NO: 38) to each 20 μl system to monitor miRNA tailing and inversion recording quality.

[0043]

[0044] (Note: The enzymes used in this experiment are all products of Beijing Kuangbo Biotechnology Co., Ltd.)

[0045] ii. Put the PCR tube containing the prepared reaction solution into a PCR machine (Thermo) and incubate at 37° C. for 1 hour.

[0046] (2) RT-PCR to obtain cDNA single strand:

[0047] i. Add 0.5 μl (0.5ng / μl) of RT-Primer (Shanghai Sangon Bioengineering Technology Co., Ltd. Synthesis), incubate at 70°C for 5 minutes, and immediately place on ice for at least 2 minutes.

[0048] ii. Pr...

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Abstract

The invention provides a hepatitis B microRNA molecular marker composition and application thereof in preparing a hepatitis B infection diagnosis and/or prognosis evaluation kit. The hepatitis B microRNA molecular marker composition comprises more than one microRNA molecule as shown in SEQ NO. 1-18. The invention also provides a diagnostic kit for instructing the infection diagnosis and/or prognosis evaluation of hepatitis B. The hepatitis B microRNA molecular marker and the diagnostic kit which are provided by the invention have the characteristics of easiness for operation, safety, no injury, high specificity, high sensitivity and easiness for large-scale screening in instructing the infection diagnosis and/or prognosis evaluation of a hepatitis B patient.

Description

technical field [0001] The present invention relates to the field of hepatitis B diagnosis and medication, in particular to a group of hepatitis B plasma microRNA molecular marker combinations, and the use of the combination for infection diagnosis and prognosis assessment of hepatitis B patients, to guide hepatitis B patients Infection diagnosis, scientific medication and drug withdrawal not only reduce the pain of patients in medication, but also reduce the waste of medical resources. 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 specificity, and currently plays an important role in the clinical diagnosis, treatment and prognosis evaluation of tumors, blood diseases, vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/68
Inventor 李宁李启靖张永宏张卫红李鹏魏颖颖石佳
Owner 北京旷博生物技术股份有限公司
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