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Hypersensitive quantitative kit for hepatitis B virus detection and detection method

A hepatitis B virus and detection method technology, applied in biochemical equipment and methods, microbe measurement/inspection, etc., can solve the problems of measurement result error, detection result PCR amplification interference, etc., and achieve high accuracy and good PCR The effects of amplification specificity and good application prospects

Pending Publication Date: 2020-09-11
中诺(杭州)基因科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for quantitative detection of HBV DNA at home and abroad are real-time fluorescent PCR methods, but its quantification needs reference standards, standard curves and internal standard corrections, and there are still certain applications for the accurate quantification of limited sample materials and low copy number HBV limit
In the extraction method of the existing detection kit, there is a lack of calibration for the missing sample during the extraction process, and there are errors in the measurement results. In addition, the existing detection method also needs to rely on the threshold (CT) of the amplification curve for quantification, which is subject to Affected by the amplification efficiency, it is necessary to rely on the standard curve to determine the accounting amount, making the detection results susceptible to interference from PCR amplification

Method used

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  • Hypersensitive quantitative kit for hepatitis B virus detection and detection method
  • Hypersensitive quantitative kit for hepatitis B virus detection and detection method
  • Hypersensitive quantitative kit for hepatitis B virus detection and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Using the above detection method to verify the negative detection of the PCR reaction system:

[0039] Configuration of PCR reaction system: prepare different systems according to the ingredients in the table below, and do 8 repetitions for each system, wherein system 1 is to detect the negative rate between primers and probes, and system 2 is to examine whether the HBV P1 probe can amplify Increase the signal of the internal reference plasmid, and system 3 is to investigate whether the Inner P probe can amplify the signal of the HBV DNA standard plasmid:

[0040]

[0041] In the table, 5*aTaqmix is ​​digital PCR reaction solution A, 20× primer probe is digital PCR reaction solution B, template DNA solution in system 1 is 11.25 μl of DdH 2 O, the template DNA solution in system 2 is 11.25 μl of internal reference plasmid containing human RNaseP gene and DdH 2 O mixed solution, the template DNA solution in system 3 is 11.25 μl of DdH2O and HBV DNA stand...

Embodiment 2

[0047] Embodiment 2: adopt above-mentioned detection method to the detection verification of the PCR reaction system containing HBV DNA standard plasmid:

[0048] Configuration of PCR reaction system: prepare different systems according to the ingredients in the table below, and do 4 repetitions for each system, in which the templates of system 1 to system 4 are standard HBV DNA plasmids diluted tenfold.

[0049]

[0050]

[0051] In the table, 5*aTaq mix is ​​digital PCR reaction solution A, 20× primer probe is digital PCR reaction solution B, template DNA solution in system 1 is a mixed solution system of 11.25 μl of DdH2O and 5E3 copies / μl of HBV DNA standard plasmid , the template DNA solution in system 2 is 11.25 μl of DdH2O and 500 copies / μl of HBV DNA standard plasmid mixed solution, the template DNA solution in system 3 is 11.25 μl of DdH2O and 50 copies / μl of HBV DNA standard plasmid mixed solution, system The template DNA solution in 4 is a mixed solution of 11...

Embodiment 3

[0057] Embodiment 3: Carry out detection verification to containing HBV plasma standard product:

[0058] Nucleic acid sample extraction: The purchased HBV DNA quantitative national standard (Conchstein, the concentration is about 5.0E+05IU / ml) was sequentially diluted ten-fold with negative plasma to 5.0E+04, 5.0E+03, 500, 50, 5IU / ml, using the magnetic bead method plasma virus DNA extraction kit developed by TIANGEN company, according to the kit instructions and automatic nucleic acid extraction equipment operation, nucleic acid extraction first add 1.0 μl internal reference plasmid to 1000 μl plasma, and then Extract, and finally use 50 μl eluent for elution, and use it as a PCR reaction template for later use;

[0059] Configuration of the PCR reaction system: Take an appropriate amount of the PCR reaction template in the first step, prepare different PCR reaction systems according to the ingredients in the following table, and do 8 repetitions for each system, and the tot...

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Abstract

The invention discloses a hypersensitive quantitative kit for hepatitis B virus detection and a detection method. The kit comprises a digital PCR reaction solution A, a digital PCR reaction solution Band an internal reference plasmid solution, wherein the digital PCR reaction solution A is a PCR reaction premix solution, the digital PCR reaction solution B is a primer probe solution, a primer probe solution comprises a HBV upstream primer, an HBV downstream primer, an HBV probe and an internal reference plasmid primer probe; the primer sequence of the HBV upstream primer is 5'-CGCAACCTCTCTTTACGCGGTC-3', the primer sequence of the HBV downstream primer is 5'-ACGGTGGTCTCCATGCGACG-3', the probe sequence of the HBV probe is 5'-FAM-TCCCGTCTGTGCCTTCTCATCTGCCGG-BHQ-3', and the probe sequence ofthe internal reference plasmid primer probe is 5'-VIC-ACATGGGAGTGGAGTGACAGG-mgb-3'; the detection method comprises the following steps of extracting a nucleic acid sample; configuring a PCR reaction system; preparing liquid drops; carrying out PCR amplification; and analyzing and determining results. The method has the advantages of accurate detection result, reduced amplification interference, high detection sensitivity, simplicity and convenience in operation and the like.

Description

【Technical field】 [0001] The invention relates to the technical field of hepatitis B virus detection, in particular to the technical field of a hypersensitive quantitative kit and detection method for hepatitis B virus detection. 【Background technique】 [0002] HBV infection is a global public health problem. According to WHO reports, about 2 billion people in the world have been infected with HBV, and about 650,000 people die of liver failure, cirrhosis and hepatocellular carcinoma (HCC) caused by HBV infection every year. . my country is a high-prevalence area of ​​hepatitis B, about 10% of the population are HBV carriers, and the number of HBV carriers exceeds 120 million. [0003] At present, the most important treatment for patients with chronic hepatitis B is antiviral therapy, and the most widely used class of drugs is nucleoside (acid) analogs (including lamivudine, adefovir dipivoxil, telbivudine, Entecavir, tenofovir disoproxil, etc.), and other drugs such as int...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/706C12Q1/6851C12Q2600/166C12Q2545/101C12Q2563/159C12Q2563/107
Inventor 李强曹丽萍徐威张辉马涛孙聪
Owner 中诺(杭州)基因科技有限责任公司