Digital PCR quantitative detecting kit for HBV (hepatitis B virus) cccDNA and application of digital PCR quantitative detecting kit for HBV cccDNA

A quantitative detection and kit technology, applied in the field of in vitro nucleic acid detection of viruses, to achieve the effects of ingenious detection design, high sensitivity and rapid detection

Active Publication Date: 2015-03-04
SHANGHAI CHROMYSKY MEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, despite this, some studies have shown that when the total HBV quantitative result is above 1×105 copies/ml, the influence of HBV rcDNA on the detecti

Method used

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  • Digital PCR quantitative detecting kit for HBV (hepatitis B virus) cccDNA and application of digital PCR quantitative detecting kit for HBV cccDNA
  • Digital PCR quantitative detecting kit for HBV (hepatitis B virus) cccDNA and application of digital PCR quantitative detecting kit for HBV cccDNA
  • Digital PCR quantitative detecting kit for HBV (hepatitis B virus) cccDNA and application of digital PCR quantitative detecting kit for HBV cccDNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of DNA in Liver Puncture Tissue

[0047] Liver tissue puncture was performed according to medical routine. Take the liver tissue obtained by 5mm puncture, cut it into pieces with ophthalmic scissors, wash with 200 μL of PBS buffer, and centrifuge at 800 rpm to remove the supernatant, a total of 3 times. Genomic DNA was extracted from the washed liver tissue using the QIAGEN genome mini-extraction kit, and the eluent was 96 μL.

[0048] The extract uses PSAD enzyme to digest HBV rcDNA, the system is as follows:

[0049] DNA extract 32μl

[0050] PSAD enzyme (10U / μl) 2.0 μl

[0051] 10× Digestion Buffer 4.0 μl

[0052] ATP (25mM) 2.0 μl

[0053] The total volume was 40 microliters and incubated for 60 minutes at 37°C followed by 10 minutes at 95°C.

Embodiment 2

[0054] Embodiment 2: Design and synthesis of HBV cccDNA and RPP40 specific primer probe

[0055] In the upstream of the DR2 region of HBV DNA, the positive strand gap region of HBV rcDNA, according to the conservation of different genotypes of HBV, the HBV-specific primer fragment P1 was designed, and a random sequence P9 (SEQ ID No. 9) was linked to its 5' end, The nucleotide sequence shown in SEQ ID No. 1 is formed after the common upstream primer P3 (SEQ ID No.3) is linked upstream of P9, and this nucleotide sequence is the HBV cccDNA specific upstream primer. The 11 nucleotide sequences at the 5' end of P1 and the 9 nucleotides at the 3' end of P9 form the HBV cccDNA-specific detection probe P5 (SEQ ID No.5), whose 5' end is labeled with FAM fluorescein, 3' end labeled MGB. In the upstream of the HBV rcDNA minus-strand nick, according to the conservation of different genotypes of HBV, the HBV-specific primer fragment P2 was designed, and its 5' end was linked with the c...

Embodiment 3

[0058] Example 3: Quantitative detection of HBV cccDNA

[0059] (1) PCR reaction system:

[0060] Prepare the digital PCR reaction system as shown in Table 3;

[0061] Table 3 HBV cccDNA digital PCR quantitative detection reaction system

[0062]

[0063] (2) Detection chip loading:

[0064] According to the requirements of the digital PCR user manual, load the prepared PCR reaction solution into the reaction chip through the chip loading hole, and then seal the reaction chip with 5.50 μL blocking solution;

[0065] (3) PCR reaction conditions:

[0066] Carry out PCR amplification by the reaction conditions shown in table 4;

[0067] Table 4 Reaction conditions for quantitative detection of HBV cccDNA digital PCR

[0068]

[0069] (4) Interpretation of digital PCR results:

[0070] Quantification of total HBV cccDNA in the reaction system = number of blue fluorescent microwells;

[0071] Quantification of the total cell number in the reaction system = 0.5×number o...

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Abstract

The invention belongs to the technical field of virus in-vitro nucleic acid detection and particularly relates to a digital PCR quantitative detecting kit for HBV (hepatitis B virus) cccDNA and an application of the digital PCR quantitative detecting kit for HBV cccDNA. A digital PCR quantitative detecting probe composition of the HBV cccDNA is firstly designed, and the kit comprises a container for detecting the probe composition and a container for a purifying reagent containing liver puncture tissue DNA. Using of the kit comprise the following steps of extracting the DNA in the liver puncture tissue, and adopting PSAD (prostate specific antigen density) enzyme to digest the HBV cccDNA; by taking a digital PCR detecting chip as a reaction container and adopting a complex fluorescent multiple PCR primer, amplifying target DNA in detecting micro pores of the digital PCR chip; and obtaining the quantitative results of the HBV cccDNA in each cell according to a fluorescence signal in each micro pore of the digital PCR detecting chip. The digital PCR quantitative detecting kit for HBV cccDNA is strong in specificity, high in sensitivity, simple and convenient and quick. HBV cccDNA and HBV rcDNA are not needed to be separated and extracted, and the defects in the conventional HBV cccDNA detection are overcome, and therefore, the digital PCR quantitative detecting kit is suitable for large-scale popularization and application.

Description

technical field [0001] The invention belongs to the technical field of virus in vitro nucleic acid detection, and in particular relates to a HBV cccDNA digital PCR quantitative detection kit and an application thereof. Background technique [0002] Chronic Hepatitis B (CHB) is a liver inflammatory disease caused by chronic infection of Hepatitis B Virus (HBV) in human liver cells, and its continuous progression can lead to liver cirrhosis, liver function failure and liver cancer. It is estimated that there are currently about 350 million HBV carriers in the world, and about 1 million people die each year from liver failure, cirrhosis or liver cancer caused by HBV infection. In 2012, the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B" jointly formulated by the Liver Disease Branch of the Chinese Medical Association and the Infectious Disease Branch of the Chinese Medical Association pointed out that there are currently about 93 million chronic HBV infect...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2563/107C12Q2563/159
Inventor 徐文胜赵书民杭小峰赵翊均缪晓辉
Owner SHANGHAI CHROMYSKY MEDICAL RES
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