Hepatitis B virus nucleic acid quantitative detection method and kit

A hepatitis B virus and nucleic acid quantification technology, applied in the field of biochemistry, can solve problems such as low sensitivity and poor detection accuracy, and achieve the effects of simple and fast operation, improved accuracy and reduced false positives

Active Publication Date: 2013-07-03
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method and kit for the quantitative detection of HBV nucleic acid, using the magnetic bead method to extract HBV and internal reference nucleic acid in 400 μl ~ 1ml of large-volume samples, and quantitatively detect through fluorescent PCR, using a competitive internal reference And the dUTP-UNG anti-pollution system can reduce the false negative and false positive of the test results, and fundamentally solve the problems of poor detection accuracy and low sensitivity of existing methods, and is suitable for application in clinical HBV nucleic acid quantitative detection

Method used

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  • Hepatitis B virus nucleic acid quantitative detection method and kit
  • Hepatitis B virus nucleic acid quantitative detection method and kit
  • Hepatitis B virus nucleic acid quantitative detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the preparation of HBV nucleic acid quantitative detection kit

[0033] According to the content of the invention and the technical scheme, the preparation process of each component of the nucleic acid extraction part, amplification part, quantitative calibrator, reference substance and internal reference in the HBV nucleic acid quantitative detection kit is as follows:

[0034] (1) Lysis buffer

[0035] Contains 50mM Tris-HCl (pH8.0), 6M guanidine hydrochloride, 1% (w / v) SDS, 10% (v / v) Triton X-100.

[0036] (2) Wash buffer W1

[0037] Contains 10 mM Tris-HCl (pH 8.0), 30% ethanol.

[0038] (3) Wash buffer W2

[0039] Contains 10 mM Tris-HCl (pH 8.0), 80% ethanol.

[0040] (4) Elution buffer

[0041] It is 10mM Tris-HCl (pH8.0) solution.

[0042] (5) Magnetic beads

[0043] (6) PCR buffer

[0044] Contains 33.33mM Tris-HCl (pH8.3), 166.67mM KCl, 0.67mM dNTPs, 10mM MgCl 2 , 0.2-0.5μM HBV primer, HBV fluorescent probe and internal reference fluores...

Embodiment 2

[0059] Embodiment 2: the determination of kit detection sensitivity

[0060] (1) Extraction of HBV DNA from serum samples

[0061] Three dilution gradients of HBV DNA positive serum with known concentrations calibrated by the National Institute for the Control of Pharmaceutical and Biological Products Hepatitis B virus (HBV) nucleic acid quantitative standards were the samples to be determined (each concentration was 1.0×10 1 IU / ml, 1.0×10 2 IU / ml, 1.0×10 3 IU / ml), measure the sensitivity of the test kit prepared in Example 1 to detect HBV DNA. Sample HBV DNA extraction steps are as follows:

[0062] ① Take the serum samples of the above three concentrations, add 800 μl each of the 4 quantitative calibrators of the kit, the critical positive control and the negative control, add 800 μl of lysis buffer, 20 μl of magnetic beads and 5 μl of the internal reference, and put them in a 2ml centrifuge tube to mix evenly , placed at room temperature for 10 minutes.

[0063] ②Magne...

Embodiment 3

[0071] Example 3: Application of the kit in the extraction and detection of serum HBV DNA

[0072] (1) Extraction of HBV DNA from serum samples

[0073] Take 5 cases of known concentrations of HBV DNA positive serum, use the kit of the present invention prepared in Example 1 to extract and detect, and select the HBV nucleic acid fluorescent quantitative PCR detection kit of Shanghai Cloning Bio-High Technology Co., Ltd. as a control for extraction and detection.

[0074] The serum HBV DNA extraction procedure of the present invention is the same as in Example 2, and the eluted nucleic acid is taken for fluorescent PCR detection. The nucleic acid extraction method of the control kit sample of Shanghai Cloning Biotech Co., Ltd. is as follows: take 50 μl of serum, add 50 μl of nucleic acid extraction solution A, shake and mix for 10 sec, centrifuge at 13,000 rpm for 10 min, discard the supernatant; add 50 μl of nucleic acid extraction solution B to During precipitation, shake an...

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Abstract

The invention relates to a hepatitis B virus (HBV) nucleic acid quantitative detection method and a kit. The method includes: employing a magnetic bead technique to extract HBV and internal reference nucleic acid from a sample to serve as a template, and finishing HBV nucleic acid quantitative detection based on a fluorescent PCR technology. The introduced internal reference can be used for monitoring the loss and misoperation of the nucleic acid template or existent PCR inhibitors during operation, thus avoiding a false-negative result. An adopted dUTP-UNG enzyme system can reduce cross contamination of amplification products and prevents a false-positive result. The kit includes a nucleic acid extraction reagent, an amplification reagent, quantitative calibration substances, reference substances and the internal reference. The method and the kit solve the shortcomings of poor detection accuracy and low sensitivity in the prior art, and are suitable for application in clinical HBV nucleic acid quantitative detection.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a quantitative detection method and kit of hepatitis B virus nucleic acid. Background technique [0002] my country is a big country with hepatitis B virus (Hepatitis B Virus, HBV) infection and transmission. An effective means of evaluating the effect of treatment. [0003] Fluorescence quantitative PCR detection of HBV nucleic acid first needs to extract the sample nucleic acid. At present, the main nucleic acid extraction methods in domestic and foreign clinical applications are: (1) Alkaline lysis and boiling method, or at the same time, polysaccharide concentration and precipitation of HBV virus is carried out first, and lysis is added to the precipitation. (2) silica gel membrane adsorption column method, using silica gel membrane chromatography column to absorb, wash, and elute to obtain nucleic acid; (3) magnetic bead adsorption method, using magnetic bea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 吴大治夏懿韩倩吴梅
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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