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Fluorescence quantitative PCR detection kit of hepatitis B virus and application thereof

A hepatitis B virus and detection kit technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of large sample demand, narrow quantitative range, and difficulty, and achieve The effect of reducing personnel operation errors, improving detection sensitivity, and improving stability

Active Publication Date: 2010-05-05
SANSURE BIOTECH
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Problems solved by technology

[0005] A variety of kits for the quantitative detection of HBV-DNA based on real-time fluorescent quantitative PCR technology have been used in clinical testing in China. The HBV-DNA extraction method provided by these kits is mainly the boiling method. This type of kit is easy to operate and fast. etc. advantages, however, there are many weak points: 1) specificity is not very good, can not detect eight genotypes (A~H) of all HBV; 2) because nucleic acid extraction process is simple, without nucleic acid purification process, can't effectively Remove PCR inhibitors in complex samples (such as high blood fat, milk, etc.), and there is generally no positive internal control (ie, internal standard) in the system, which cannot prevent false negatives; 3) The detection sensitivity is low, about 500IU / ml ; The quantitative range is narrow, generally between 1.00E+03IU / ml and 1.00E+08IU / ml, for samples with clinical high values ​​(greater than 1.00E+08IU / ml) and low values ​​(less than 1.00E+03IU / ml) Unable to accurately quantify; 4) Generally, there are no measures to prevent PCR product contamination; 5) There is no fluorescence normalization correction system
The diagnostic system has the advantages of high degree of automation, easy operation, and low detection sensitivity (greater than 12IU / ml), but the sample requirement (1ml) is too large, and the cost of reagents and consumables for fully automated operation is too high, so it is difficult to apply in clinical practice. Widely carried out

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  • Fluorescence quantitative PCR detection kit of hepatitis B virus and application thereof
  • Fluorescence quantitative PCR detection kit of hepatitis B virus and application thereof
  • Fluorescence quantitative PCR detection kit of hepatitis B virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: specificity test

[0051] The known HBV-positive samples and HBV-negative samples were used for detection to examine the specificity of the kit. The formulations and operating procedures of each reagent are as follows:

[0052] Reagent preparation:

[0053] DNA extraction solution I: containing sodium lauryl sulfate 0.2% (mass / volume), triton 1.0% (volume / volume), guanidine isothiocyanate 0.2mol / L;

[0054] DNA extraction solution II: containing 4-hydroxyethylpiperazineethanesulfonic acid 100mmol / L, pH6.3, sodium chloride 100mmol / L, magnetic beads 100μg / ml;

[0055] DNA extraction solution III: containing Triton 0.1% (volume / volume), sodium chloride 100mmol / L;

[0056] DNA extraction solution IV: mineral oil;

[0057] The internal standard (positive internal control) is: a recombinant of a 97-base-pair artificially synthesized DNA sequence inserted into the pUC18T vector, that is, a plasmid (hereinafter referred to as a recombinant plasmid), with a con...

Embodiment 2

[0085] Embodiment 2: The impact of different extraction methods of DNA on the detection of HBV

[0086] Existing a lot of mature DNA extraction kits on the market can be used for the extraction of HBV-DNA in the sample at present, select common boiling cracking method and column passing method to compare with the magnetic bead method extraction nucleic acid in the kit of the present invention, select high, Eight cases of HBV-positive samples (including serum, plasma and breast milk) with medium and low concentrations were subjected to fluorescent PCR reaction after extracting DNA respectively. The reagent formula and detection steps are as follows:

[0087] Reagent preparation:

[0088] The formula of DNA extraction solution I: sodium lauryl sulfate 0.5%, triton 2.5%, guanidine isothiocyanate 0.6mol / L;

[0089] The formula of DNA extraction solution II: 4-hydroxyethylpiperazineethanesulfonic acid 200mmol / L, pH6.5, sodium chloride 200mmol / L, magnetic beads 250μg / ml;

[0090]...

Embodiment 3

[0135] Example 3 Fluorescence normalization correction

[0136] Due to the unavoidable accidental factors such as the error of sample loading operation, the difference in the light transmission performance of the centrifuge tube, and the difference in the fluorescence excitation efficiency, the original signal collected by the instrument must be normalized to eliminate the influence of these factors on the test results. This correction can be achieved by adding additional fluorescent dyes (called reference fluorescence) in the reaction system, such as adding ROX reference fluorescence, the concentration of ROX in the PCR reaction solution is constant, so the intensity of its signal is related to the reaction The volume of the system is positively correlated with the fluorescence excitation efficiency. When the reaction volume changes or the excitation light efficiency changes, the ROX signal and the signal of the target gene (such as FAM) are affected to the same degree (assumi...

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Abstract

The invention discloses a fluorescence quantitative PCR detection kit of hepatitis B virus and an application thereof. The kit is composed of the following independent components: DNA extraction solution I, DNA extraction solution II, DNA extraction solution III, DNA extraction solution IV, positive control interior label, PCR reaction liquid, probe HBV-SP, enzyme mixed liquor containing heat resistant DNA polyase and uracil DNA glycosylase, quantitative hepatitis B virus reference material, hepatitis B virus positive control serum and hepatitis B virus negative control serum, wherein DNA extraction solution I contains 0.2-1.0% of lauryl sodium sulphate (mass / volume), 1.0-4.0% of Triton (volume / volume) and 0.2-1.0mol / L of guanidinium isothiocyanate; DNA extraction solution II contains 100-300mmol / L of 4-HEPES, 100-300mmol / L of sodium chloride with pH of 6.5+ / -0.2 and 100-400 mu g / ml of magnetic beads; DNA extraction solution III contains 0.1-1.0% of Triton (volume / volume) and 100-300mmol / L of sodium chloride; DNA extraction solution IV contains mineral oil. The fluorescence quantitative PCR detection kit of hepatitis B virus of the invention can be used for detecting the HBV-DNA concentration in samples of serum, blood plasma or latex and the like.

Description

technical field [0001] The invention relates to a hepatitis B virus fluorescence quantitative PCR detection kit and its application, in particular to a hepatitis B virus rapid fluorescence quantitative PCR detection kit and its application in suspicious samples such as serum, blood plasma and milk. Background technique [0002] Viral hepatitis B (hepatitis B) is an infectious disease caused by hepatitis B virus (Hepatitis B Virus, HBV), which mainly causes liver lesions and can cause damage to various organs. A small number of patients with hepatitis can develop cirrhosis or liver cancer. Chronic hepatitis B virus infection is a health problem that plagues the world. There are about 350 million patients with chronic hepatitis B infection in the world, and the number of deaths caused by chronic hepatitis B virus is as high as 1.2 million every year. It is listed as one of the nine leading causes of death in the world. China is a big country with hepatitis B. There are about ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 戴立忠邓中平熊晓燕
Owner SANSURE BIOTECH
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