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Serum miRNA composition for detecting occult hepatitis B and applications thereof

A hepatitis B, composition technology, applied in the field of serum miRNA composition, to achieve the effect of easy acquisition, convenient detection, and low storage conditions

Inactive Publication Date: 2013-02-27
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent studies have shown that serum / plasma microRNA can be used for the early diagnosis and treatment guidance of various cancers, but there is no report on the use of serum / plasma microRNA in the detection of occult hepatitis B

Method used

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  • Serum miRNA composition for detecting occult hepatitis B and applications thereof
  • Serum miRNA composition for detecting occult hepatitis B and applications thereof
  • Serum miRNA composition for detecting occult hepatitis B and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The collection of embodiment 1 sample and the arrangement of sample data

[0038]The inventor collected a large amount of serum samples from the Nanjing Second Hospital between October 2009 and May 2010. By sorting out the sample data, the inventor selected 30 healthy controls (average age: 35.60) ±15.27, age span: 20-61, male: 14, female: 16), 29 patients with chronic hepatitis B (average age: 39.24±12.83, age span: 14-67, male: 17, female: 12), Eleven patients with occult hepatitis B (average age: 42.82±17.96, age span: 23-80, male: 5, female: 6) were used as experimental samples for real-time PCR verification. Specific sample classification criteria are as follows:

[0039] Group CTL: healthy control group (n=30)

[0040] (1) Hepatitis B surface antibody, hepatitis B surface antigen and hepatitis B core antibody negative

[0041] (2) ALT<40IU / L, AST<45IU / L

[0042] (3) HBV-DNA<500copies / ml

[0043] (4) No other systemic diseases

[0044] (5) Age ≥ 20

[0045] ...

Embodiment 2

[0057] Example 2 Preliminary screening of specific miRNA expression profiles in patients with chronic hepatitis B

[0058] Using Solexa sequencing technology to discover and prove that there are 88 differentially expressed microRNAs in the serum / plasma of 30 healthy controls and 29 chronic hepatitis B patients. The specific steps are:

[0059] (1) Collect serum / plasma from healthy controls and chronic hepatitis B patients;

[0060] (2) Take 80-100ml of serum respectively and add an equal volume of Trizol Reagent;

[0061] (3) Phase separation: place at room temperature for 15 minutes, then add chloroform according to the volume ratio of 0.2ml chloroform / 1ml Trizol Reagent, shake for 15s in the education column, 15 minutes at room temperature, 12,000g, 4°C, centrifuge for 15 minutes;

[0062] (4) Transfer the aqueous phase to a new 50ml centrifuge tube, and remove the protein phase in 3 steps of phenol / chloroform;

[0063] (5) RNA precipitation: transfer the aqueous phase to...

Embodiment 3

[0076] Example 3 Re-screening with quantitative PCR method and evaluating the clinical value of serum microRNA in OBI detection

[0077] The qRT-PCR test of microRNA was performed on the sera of 30 healthy controls, 29 patients with chronic hepatitis B, and 11 patients with occult hepatitis B with the probe.

[0078] (1) Extract serum total RNA by phenol / chloroform method

[0079] a) Take 100ul sample and add it to 300ul water, mix well, add 200ul (PH=4.7-5.5) acidic phenol, shake vigorously and mix well, add 200ul chloroform after standing at room temperature for 2 minutes, shake fully again, let stand at room temperature for 5 minutes, then 12000g Centrifuge at room temperature for 15 minutes;

[0080] b) Carefully draw the supernatant (about 400 ul) into 800 ul of isopropanol, then add 40 ul of 3M sodium acetate with pH=5.2, mix well, let stand at -20°C for 30 minutes, then centrifuge at 16000g for 20 minutes at 4°C;

[0081] c) After fully discarding the supernatant, add...

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Abstract

The invention belongs to the field of biotechnology, and discloses a serum miRNA composition for detecting occult hepatitis B and applications thereof. The serum miRNA composition for detecting occult hepatitis B is selected from at least two of miRNA:hsa-let-7c, hsa-miR-23b, hsa-miR-122 or hsa-miR-150. The invention also provides a TaqMan probe composition for detecting the occult hepatitis B, which comprises a TaqMan probe of the miRNA described in the invention. The miRNA composition can be used for preparing a reagent or tool for detecting OBI (occult hepatitis B virus infection). Through the development and application of serum microRNA biomarkers and test kits, the detection of the OBI is convenient and easy to implement, thereby providing a support for clinicians to quickly detect occult hepatitis B patients and timely take effective prevention and treatment schemes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a serum miRNA composition for detecting occult hepatitis B and an application thereof. Background technique [0002] Occult HBV infection (OBI) refers to negative serum HBsAg, but positive HBV-DNA in serum and (or) liver tissue, and clinical manifestations of chronic hepatitis B. -HBs, anti-HBe and (or) anti-HBc positive, about 20% of occult hepatitis B patients except HBV-DNA positive, the rest of the HBV serological markers are negative. OBI is associated with many clinical situations and is a complex epidemiological and clinical problem because it is difficult to detect by conventional methods. [0003] The cause of OBI is still not very clear, it may be related to the following factors. (1) Low-level replication of HBV, (2) HBV gene variation, (3) HBV integration with host chromosome, (4) HBsAg expression and secretion are inhibited, (5) abnormal host immune response, (6) sensiti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 曾科陈渊源李丽民张辰宇
Owner NANJING UNIV
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