Quantitative detection kit, detection method, primer and probe for hepatitis B virus nucleic acid

A hepatitis B virus, detection kit technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Time and other issues, to achieve high specificity, simple technical operation, and reliable results

Active Publication Date: 2011-08-10
WUHAN BIOTECH GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These PCR-based detection methods all need time to post-process the PCR amplification products. The high concentration of target DNA molecules in the PCR amplification products will pollute the entire experimental space in the form of aerosols. Due to the high sensitivity of PCR, It can even detect the template concentration of 10 copies in the reaction system, which may bring serious false positives to future PCR experiments; the conventional PCR method is an end-point detection method, because the PCR reaction has a plateau period, it is impossible to base the final product on the starting point. template for accurate quantitative detection

Method used

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  • Quantitative detection kit, detection method, primer and probe for hepatitis B virus nucleic acid
  • Quantitative detection kit, detection method, primer and probe for hepatitis B virus nucleic acid
  • Quantitative detection kit, detection method, primer and probe for hepatitis B virus nucleic acid

Examples

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Effect test

Embodiment 1

[0109] Method for detecting hepatitis B virus nucleic acid on ABI 7300 fluorescent quantitative PCR instrument with kit of the present invention

[0110] (1) Collect samples: collect human serum samples;

[0111] a. Use a sterile syringe to extract 1ml of venous blood from the subject, inject it into a sterile 1.5ml centrifuge tube, let it stand at room temperature for 2 hours, transfer it to 4°C for 1 hour, centrifuge at 8000rpm for 5 minutes, and draw 200μL of supernatant (note: Do not inhale red blood cells), transfer to another sterile 1.5ml centrifuge tube, which is the serum sample.

[0112] b. Specimen storage and transportation: If the specimen is not tested immediately, it should be stored at -20°C and avoid repeated freezing and thawing. Long-distance transportation of specimens should use 0 ℃ curling.

[0113] (2) Sample treatment: Take 100 μL of the serum to be tested, add an equal amount of 15% solution A, mix well, centrifuge at 13000 rpm for 10 minutes, remove...

Embodiment 2

[0126] Use the kit of the present invention to detect the hepatitis B virus nucleic acid of the unknown sample according to the method of Example 1. Unknown sample source ×× hospital patient's sample to be measured, the test result of embodiment 2 of the present invention is as follows figure 2 As shown, the test results are shown in the table below:

[0127] serial number

[0128] 2

[0129] Among them, the samples are all within the positive range and are positive samples.

Embodiment 3

[0131] Detect the hepatitis B virus nucleic acid of unknown sample with kit of the present invention, except sample processing method, all the other are basically the same with the method of embodiment 1, the sample processing method of the present embodiment is as follows:

[0132] Sample treatment: 50 μL of 0.4M NaOH was added to 50 μL of serum, and incubated at 80° C. for 10 min. Centrifuge at 15000r for 30s, take the supernatant, add 25 μL of 0.4M Tris.HCl pH7.5, and store at -4°C for later use.

[0133] The test result of the embodiment of the present invention 3 is as image 3 As shown, the test results are shown in the table below:

[0134] serial number

[0135] Among them, the samples are all within the positive range and are positive samples.

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Abstract

The invention discloses a quantitative detection kit, detection method, primer and probe for hepatitis B virus nucleic acid, belonging to the technical field of biology. The invention relates to a gene detection technology of a virus causing various types of acute, chronic and severe hepatitis of human beings, which is suitable for qualitative and quantitative detection of the hepatitis B virus. The kit comprises PCR (Polymerase Chain Reaction) reaction liquid, wherein the reaction liquid comprises DEPC (Diethyl Pyrocarbonate) treating water, Taq enzyme, dNTPs (Deoxynucleotide Triphosphates),10*PCR Buffer, a solution containing Mg<2+> ions, a hepatitis B virus positive primer: 5'-TTGTCCTGGYTATCGYTGGAT-3', a hepatitis B virus negative primer: 5'-TGAGGCATAGCAGCAGGATGA-3', a hepatitis B virus probe: 5'-CTGCGGCGTTTTAT-3'; and the kit also comprises a DNA (Deoxyribonucleic Acid) extracting solution, a negative control material, a working standard product, a positive control material and acritical positive control material. The hepatitis B virus nucleic acid is detected quantificationally by adopting a real-time fluorescent quantitative PCR technology. The invention has the characteristics of specificity, sensitivity, quickness and easiness and convenience for operation.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a hepatitis B virus nucleic acid quantitative detection kit and a detection method thereof. Background technique [0002] Hepatitis B virus (hepatitis B virus, hepatitis B virus) belongs to hepadnaviridae, and its genome is about 3.2kb in length, which is partially double-stranded circular DNA. HBV replication is characterized by the presence of stable covalently closed circular DNA (cccDNA) in the nucleus of hepatocytes and a reverse transcription step. There are 4 identified open reading frames in the hepatitis B virus genome, which encode respectively the nucleocapsid (C) and envelope (S) proteins of the virus, the viral replicase (polymerase) and a protein that seems to be related to viral gene expression. Protein X. Hepatitis B virus is the smallest DNA virus found so far. Hepatitis B virus is the main pathogenic factor of acute and chronic viral hepatitis, w...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12N15/11C12Q1/68G01N21/64
Inventor 唐景峰柳小英王业富严晓峰白旭
Owner WUHAN BIOTECH GENE ENG
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