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Method and kit for detecting HBV PgRNA in blood of patient with hepatitis b and application thereof

A hepatitis B, kit technology, applied in the field of molecular biology, can solve problems such as unrealizable, and achieve the effects of convenient use, reliable detection results, and high detection sensitivity

Inactive Publication Date: 2016-08-31
北京旌准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The main problem solved by this application is to provide methods and kits for detecting HBV PgRNA in the blood of patients with hepatitis B, so as to solve the technical problems that cannot be realized

Method used

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  • Method and kit for detecting HBV PgRNA in blood of patient with hepatitis b and application thereof
  • Method and kit for detecting HBV PgRNA in blood of patient with hepatitis b and application thereof
  • Method and kit for detecting HBV PgRNA in blood of patient with hepatitis b and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] 1. Sample collection: collect serum or plasma.

[0088] 1) Serum sample collection: Use a sterile syringe to draw 3-5 mL of venous blood from the bend of the subject’s arm or the back of the hand, inject it into a sterile collection tube, and leave it at room temperature for no more than 4 hours until the sample precipitates serum by itself or directly centrifuge at 1600 rpm at room temperature After 5 minutes, the serum was separated and transferred to a 1.5mL sterilized centrifuge tube for later use.

[0089] 2) Plasma sample collection: Use a sterile syringe to draw 3-5 mL of venous blood from the bend of the subject's arm or the back of the hand, inject it into a sterile collection tube containing EDTA, immediately invert and mix it evenly, and place it at room temperature for no more than 4 hours. Wait for the sample to precipitate the plasma by itself or directly centrifuge at room temperature at 1600rpm for 5 minutes to separate the plasma, and transfer it to a 1...

Embodiment 2

[0132] Design of primers and probes:

[0133] At present, the HBV genotypes in my country are mainly B and C, with a small amount of D, and a small amount of fusion types of the above genotypes.

[0134] In the present invention, when designing primers and probes, B-type, C-type and D-type are taken into account. Referring to the sequences of all HBV types B, C and D in the internationally recognized database NCBI, after comparison, the sequence part of PgRNA that is different from other mRNAs, that is, the part between about 1800bp and 2800bp, was selected for analysis. The designed primers and probes are located in the conserved or relatively conserved region sequences of each genotype of HBV, ensuring the compatibility of the "primers and probes used in the patent" to various common genotypes of HBV in China. Primers with base sequences shown in SEQ ID NO: 1-NO: 6 and probes with base sequences shown in SEQ ID NO: 7 were designed.

[0135] Screening of primers and probes:...

Embodiment 3

[0146] DNA Amplification Verification of HBV pgRNA Extract

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Abstract

The invention discloses a method for detecting HBV PgRNA in blood of a patient with hepatitis b. After pgRNA is extracted, the extracted HBV pgRNA is purified by RNase-free Dnase I; and quantitative determination on pgRNA is achieved by adopting the qRT-PCR method. The invention further provides a kit for detecting the HBV PgRNA in blood of the patient with hepatitis b. The method has the following effects: firstly, the method adopts blood detection, is noninvasive, is normal and is capable of being applied on a large scale; secondly, PgRNA with ultralow carrying capacity in the blood can be detected; and thirdly, the kit and the application thereof in predicating NA drug withdrawal can be used for researching the shape of Chinese PgRNA shape, so that fundamental research data is provided for developing a PgRNA drug. A scheme is provided for solving the problem of finding clinical criteria and a biological marker for predicating NA drug withdrawal, which is one of ten to-be-solved problems of hepatitis B clinical guideline.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method, kit and application for detecting HBVPgRNA in the blood of hepatitis B patients. Background technique [0002] The life process of hepatitis B virus particles in the human body has been thoroughly studied: there is a reverse transcription step in the replication process of HBV, so its life cycle is different from other DNA viruses. Due to the characteristics of reverse transcriptase, the mutation rate of HBV is much higher . [0003] HBV DNA exists in the form of relaxed circular DNA (rcDNA) in the complete virus. After the HBV virus particle enters the liver cell, it takes off the outer capsid, and its rcDNA enters the nucleus to form a covalently closed circular DNA ( covalentlt closed circular DNA, cccDNA), using cccDNA as a template, transcribed to form pre-genomic RNA (PgRNA) and other mRNAs, PgRNA forms rcDNA under the action of reverse transcriptase, r...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/706C12Q1/6851C12Q2600/118C12Q2600/158C12Q2531/113C12Q2545/113
Inventor 叶锋刘明坤余荣
Owner 北京旌准医疗科技有限公司
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