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Homogeneous molecular lampmark fluorescent probe PCR hepatitis B virus detecting method and reagent kit

A detection method and homogeneous fluorescence technology, applied in biological testing, biochemical equipment and methods, material testing products, etc., can solve the problems of hepatitis B detection research and application that have not yet been reported, cannot be eliminated, time-consuming and labor-intensive, etc., to avoid The use of external references, easy operation, and the effect of avoiding cumbersome operations

Inactive Publication Date: 2003-11-05
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this invention provides a relatively simple nucleic acid molecular detection method, it still cannot get rid of the sample spotting-hybridization-enzyme labeling-color development required for heterogeneous hybridization detection to perform a series of cumbersome operations, which are time-consuming, laborious and very inconvenient
[0005] Molecular beacon probes have been successfully applied in the detection of Mycobacterium tuberculosis (CN1281901A) and intracellular gene analysis (CN1308135A), but the application in the detection of hepatitis B has not been reported yet

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] In this embodiment, when the PCR reaction solution contains different Mg 2+ The detection effect of the molecular light marker at the concentration can determine the Mg in the PCR reaction solution 2+ concentration.

[0027] A series of PCR amplification reactions were carried out with normal human serum and human serum containing hepatitis B virus as negative and positive templates (see the following examples for the processing method of serum samples). Wherein the concentration of Mg2+ rises from 0mmol / L to 10mmol / L with a gradient of 0.5mmol / L, and other PCR reaction components are as follows: 1×PCR reaction buffer (10mmol / LKCl, 8mmol / L (NH 4 ) 2 SO 4 , 10mmol / L Tris-HCl, pH9.0, NP-40), 1.5mmol / L MgCl 2 , each 0.4mmol / L dATP, dTTP, dCTP, dGTP, each 0.4μmol / L upstream and downstream primers (see the example below), 2.5U Taq DNA polymerase, 0.3μmol / L molecular beacon probe, 2μl template.

[0028] Carry out PCR amplification according to the following conditions: ...

Embodiment 2

[0032]The present invention is prepared following kit:

[0033] Its raw materials are selected as follows:

[0034] Molecular beacon probe: The target sequence of the probe is located in the conserved region of the hepatitis B virus gene sequence. The probe is suitable for four subtypes of hepatitis B (adr, adw, ayr, ayw), and has typical temperature denaturation and Refolding characteristics and good specificity. The 5' end was labeled with FAM and the 3' end was labeled with DABCYL.

[0035] Its sequence is:

[0036] FAM-5'-CGAGCATCTTCTGCGACGCGGGCTCG-3'-DABCY was commissioned to synthesize by Shanghai Shenyou Biotechnology Co., Ltd.

[0037] PCR primers: the amplified fragment contains the target sequence of the molecular light probe, the length of the amplified fragment is 180bp, synthesized by Dalian Bao Biological Engineering Co., Ltd., the sequence is:

[0038] Upstream primer: 5'-CACCAAATGCCCCTATCTTA-3'

[0039] Downstream primer: 5'-GTTTCCACCTTATGAGTCC-3'.

[004...

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Abstract

The invention is a method for detecting hepatitis B virus by PCR with a homogeneous molecular lamp fluorescent probe. According to the hepatitis B virus gene sequence, design specific molecular light probes in its conserved region, use polymerase chain reaction (PCR) to amplify the specific fragments of hepatitis B virus genes, use molecular light probes to detect The amplified product is detected. The detection process can be completed by directly measuring the fluorescence of the reaction system. According to the fluorescence intensity of the system, it is determined whether there is hepatitis B virus in the sample. This method can detect more than 30 copies of the virus in the serum. It exists and is easy to operate, and is suitable for the simultaneous detection of a large number of serum samples. The invention also provides a kit for applying the method.

Description

technical field [0001] The invention relates to a PCR detection method of hepatitis B virus, which uses a molecular beacon probe to detect the PCR amplification product of hepatitis B virus DNA. The invention relates to a kit for detecting the PCR amplification product of hepatitis B virus DNA by using a molecular beacon probe. Background technique [0002] Hepatitis B virus infection is a worldwide infectious disease that can cause extreme debilitation and even death in patients. It is estimated that about 200 million people in the world are carriers of hepatitis B virus, and there are 100 million people in my country, which is a high prevalence area of ​​hepatitis B and a country with a high incidence of liver cancer. Therefore, it is necessary to study the molecular biological characteristics of hepatitis B virus It is very necessary to use modern molecular biology techniques to explore diagnosis and prevention methods. [0003] The diagnosis of hepatitis B virus infecti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12Q1/25G01N33/50
Inventor 沈含熙孔德明古珑
Owner NANKAI UNIV
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