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Peripheral blood detection method of hepatitis B virus integration in liver

A hepatitis B virus, peripheral blood technology, applied in biochemical equipment and methods, microbial determination/examination, etc., can solve problems such as limited information, no research to explore specificity and sensitivity, etc., to avoid complications, and to have good specificity. , the effect of avoiding heterogeneity bias

Active Publication Date: 2020-02-28
张大可
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the information that can be provided by using cfDNA to detect mutations and methylation is still very limited, and it is necessary to explore means to provide more information
Virus integration is a characteristic event in the genome of hepatocytes after HBV infection. At present, there is no research at home and abroad to explore whether the DNA fragment at the integration site can be released into the peripheral blood, let alone whether it has sufficient specificity and sensitivity. for detection analysis

Method used

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  • Peripheral blood detection method of hepatitis B virus integration in liver
  • Peripheral blood detection method of hepatitis B virus integration in liver
  • Peripheral blood detection method of hepatitis B virus integration in liver

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1, sample collection and extraction of peripheral blood free DNA

[0077] Peripheral blood samples of 7 HCC patients after HBV infection were collected in Beijing You'an Hospital. For each liver cancer patient, in addition to collecting peripheral blood samples before surgery, 2 liver cancer tissue samples and 2 para-cancerous liver tissue samples were collected after surgery to control the ability to detect viral integration events in peripheral blood . The above-mentioned patients included 3 females and 4 males, with an average age of 51 years.

[0078]cfDNA from peripheral blood samples and DNA from liver cancer tissue samples and paracancerous liver tissue samples were extracted. DNA was extracted using the QIAGEN DNeasy Blood&Tissue Kits kit, and extracted according to the standardized procedure of the kit. The following steps are used for the extraction of cfDNA, with quantitative and quality assessments:

[0079] 1) Use free DNA blood collection tubes...

Embodiment 2

[0084] Example 2, End Repair, Adapter Ligation and Preamplification of Peripheral Blood Free DNA

[0085] The cfDNA obtained in Example 1 was processed according to the following steps:

[0086] 1) Quantify the extracted cfDNA to a volume of 50ul with ultrapure water, add 7ul of End Repair & A-Tailing Buffer and 3ul of End Repair & A-Tailing Enzyme Mix, mix to 60ul, Complete the end repair reaction on a thermal cycler, the reaction conditions are: 20°C for 30 minutes, 65°C for 30 minutes, store at 4°C, and keep the heating lid at 70°C.

[0087] 2) Add the above 60ul products to 5ul adapters (the adapters include primer regions for subsequent amplification and individual tag recognition sequences, which correspond to the adapters required by the subsequent sequencing platform), and perform ligation reaction on ice: add 55ul ligation reaction mixture , including 5ul nuclease-free water, 30ul ligation buffer, 10ul DNA ligase. Incubate for 15 minutes at 20°C (without heated lid)...

Embodiment 3

[0092] Embodiment 3, hybrid capture experiment

[0093] The integration event capture of peripheral blood cfDNA and liver cancer tissue and paracancerous tissue DNA is carried out as follows:

[0094] 1) Design of virus integration capture probe set: According to the sequences of hepatitis B virus A-H types obtained in the NCBI database, including 212 pieces of B type and 457 pieces of C type that are prevalent in China, design a degeneracy covering 3.2K of the whole virus genome Probes, each probe is about 100bp in length. The design method of the probe is well known in the art, and it can be completed by using a conventional design method, such as common software or a website. The probe set in this example is specifically designed through a commonly used primer design website https: / / design.igenetech.com / design obtained.

[0095] 2) Prepare the reaction solution: take the peripheral blood cfDNA dry powder sample obtained in Example 2, add nuclease-free water to quanti...

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Abstract

The invention provides a method for detecting the hepatitis B virus integration condition, which is used for detecting by taking free DNA in peripheral blood as a sample. According to the method, therisk of complications caused by traditional methods, such as surgical biopsy, aspiration biopsy and the like can be avoided, the safety can be improved, periodical monitoring with high frequency can be realized, and heterogeneity bias of tissue sampling can be avoided. The method has good specificity and high sensitivity, can detect a fragment in a peripheral blood sample of a patient with liver cancer after 100 percent of HBV infection, and minimally detect a fragment in all cfDNAs extracted from the plasma of 5ml of peripheral blood.

Description

technical field [0001] The invention relates to the detection of the integration status of the hepatitis B virus, in particular to the application of free DNA in peripheral blood in the detection of the integration status of the hepatitis B virus. Background technique [0002] Hepatitis B Virus (HBV), as a retrovirus, has the ability to integrate into the host genome. It was first reported in the early 1980s, and there are many studies on liver cancer genomes. 1-13 . The integration ability of HBV is not necessary for its life cycle (Life Cycle) and infecting the host. Currently, there is no gene encoding a viral protein with integrase activity in the hepatovirus genome 14 , earlier studies had considered HBV integration events to be rare events distributed randomly across the genome 15 . Studies on the integration mechanism of HBV suggest that the main form of virus integration into host genes is linear double-stranded DNA (double strand linear DNA, dsL DNA) 16 , is an ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6869
CPCC12Q1/6869C12Q1/6886C12Q2600/118C12Q2600/156C12Q2525/191
Inventor 张大可
Owner 张大可
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