A hbv DNA digital PCR quantitative detection kit and its application

A quantitative detection and kit technology, applied in the field of HBV DNA digital PCR quantitative detection kits, can solve the problem of fluorescence signal intensity dissimilation and other problems

Active Publication Date: 2017-10-20
SHANGHAI CHROMYSKY MEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another problem brought about by this is that the relationship between the fluorescence signal intensity and the amount of HBV DNA will inevitably be dissimilated.

Method used

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  • A hbv DNA digital PCR quantitative detection kit and its application
  • A hbv DNA digital PCR quantitative detection kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: DNA extraction from human plasma

[0076] Collect 2ml of human peripheral venous blood according to medical practice, EDTA anticoagulation, and centrifuge at 800rpm for 5 minutes to separate human plasma. Take 600 microliters of human plasma and extract genomic DNA with QIAGEN DNA virus extraction kit. The elution volume is 30 microliters.

Embodiment 2

[0077] Example 2: Design and synthesis of HBV DNA and RPP40 specific primer probes

[0078] Design HBV DNA specific primers and detection probes according to the three different conservative region sequences of different genotypes of HBV. The HBV specific detection probes in the three different PCR reactions are all labeled with FAM fluorescein at the 5'end. The'end is marked with MGB. RPP40-specific primers and detection probes are designed in the conservative region of the human genome RPP40 gene. The 5'-end of the RPP40-specific detection probe is labeled with VIC and the 3'-end is labeled with MGB. According to the designed three HBV specific probes and one RPP40 specific probe, the corresponding competitive probes were designed respectively. The 5'end of the competitive probe is not modified, and the 3'end is dideoxynucleotide. HBV and RPP40 specific primers and detection probes and the sequence and modification characteristics of the corresponding competitive probes are s...

Embodiment 3

[0081] Example 3: HBV DNA digital PCR quantitative detection

[0082] (1) PCR reaction system:

[0083] Prepare the digital PCR reaction system according to Table 2.

[0084] Table 2 Digital PCR reaction system for quantitative detection of HBV DNA in liver puncture tissue

[0085]

[0086] (2) Test chip loading:

[0087] Load the prepared PCR reaction solution to the detection chip through the sample hole of the chip according to the requirements of the digital PCR user manual, and then use 5.50 μL of blocking solution to seal the reaction chip.

[0088] (3) PCR reaction conditions:

[0089] Perform PCR amplification according to the reaction conditions in Table 3.

[0090] Table 3 HBV DNA digital PCR quantitative detection reaction conditions

[0091]

[0092] (4) Interpretation of digital PCR results:

[0093] Quantitative total HBV in the reaction system = number of blue fluorescent micropores

[0094] The 95% confidence interval of the above quantitative results is estimated using Possio...

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Abstract

The invention belongs to the technical field of virus external detection and in particular relates to an HBV DNA digital PCR quantitative detection kit and application thereof. The HBV DNA digital PCR quantitative detection kit comprises PCR reaction primers SEQIDNo.1 and SEQIDNo.3 and a probe SEQIDNo.2; PCR reaction primers SEQIDNo.4 and SEQIDNo.6 and a probe SEQIDNo.5; PCR reaction primers SEQIDNo.7 and SEQIDNo.9 and a probe SEQIDNo.8; reference reaction primers SEQIDNo.10 and SEQIDNo.12 and a probe SEQIDNo.11; and competitive probes SEQIDNo.13- SEQIDNo.16. When the detection kit is used, DNA of a human plasma sample is extracted, targeted DNA is amplified by adopting a composite fluorescence multiplex PCR technology in a digital PCR chip micropore, and the amount of the micropore which displays blue fluorescence is the HBV DNA quantitative result in the total reaction system. The HBV DNA digital PCR quantitative detection kit used for quantitative detection is rapid, convenient and simple, is high in sensitivity and strong in specificity and is suitable for large-scale popularization and application.

Description

Technical field [0001] The invention belongs to the technical field of virus in vitro detection, and specifically relates to a HBV DNA digital PCR quantitative detection kit and its application. technical background [0002] Chronic Hepatitis B (CHB) is an inflammatory disease of the liver caused by chronic hepatitis B virus (Hepatitis BVirus, HBV) infection in human liver cells. Its continuous progression can lead to cirrhosis and liver failure And liver cancer. It is estimated that there are about 350 million HBV carriers in the world, and about 1 million people die every 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 Diseases Branch of the Chinese Medical Association pointed out that there are currently approximately 93 million chronic HBV infections in my country, of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6851C12Q1/706C12Q2600/112C12Q2563/107
Inventor 赵书民赵翊均周巍
Owner SHANGHAI CHROMYSKY MEDICAL RES
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