Primer arm TaqMan-MGB probe PCR hepatitis b virus detection method and reagent box thereof

A hepatitis B virus and detection method technology, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems of increasing design difficulty and hindering probe combination, so as to improve efficiency, Effect of improving detection sensitivity, typical temperature denaturation and renaturation characteristics

Inactive Publication Date: 2008-12-17
淮安市第四人民医院
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the combination of TaqMan-MGB probes on the sequence to be tested makes the design of TaqMan-MGB probes for highly variable gene sequences more difficult, and it is easy to cause the protein to entangle the gene chain and hinder the binding of the probe.

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  • Primer arm TaqMan-MGB probe PCR hepatitis b virus detection method and reagent box thereof
  • Primer arm TaqMan-MGB probe PCR hepatitis b virus detection method and reagent box thereof
  • Primer arm TaqMan-MGB probe PCR hepatitis b virus detection method and reagent box thereof

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Embodiment Construction

[0029] Below in conjunction with specific embodiment, further elaborate the present invention in detail, the prominent substantive characteristic and positive effect of the present invention can be manifested from the following embodiment, but they are not to limit the present invention in any way.

[0030] Material:

[0031] (1) Bacterial strain: Escherichia coli DH5α was donated by the Department of Biochemistry, Xuzhou Medical College

[0032] (2) Reagents and solutions: Taq enzyme (Promega), vector pUC19, IPTG and X-gal, 100bp DNA Ladder Marker, PCR product recovery kit (Shanghai Gaming Company), T4 DNA ligase, restriction endonuclease BamH I And EcoR I (SibEnzyme Company), dNTP, TRIS, EDTA, Trizol, RNase Inhibitor, pUC19DNA / Mspl Marker, DEPC, tryptone for bacterial culture, yeast extract for bacterial culture (Shanghai Bioengineering Company), HBV DNA fluorescence quantification Detection Kit (Guangzhou Daan Company), Primer Synthesis (Shanghai Gaming Company), Probe Syn...

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Abstract

The invention discloses a detecting method for hepatitis B virus by PCR and using a TaqMan-MGB probe of a primer arm and a kit. A down-stream primer is added with an arm sequence; the TaqMan-MGB probe is combined with the arm sequence; the down-stream primer combined with the TaqMan-MGB probe is combined with a template and extends under the effect of Tag enzyme; an up-stream primer is combined with a product of the first circulation and extends under the effect of Tag enzyme; when the up-stream primer extends to the hybridization position of the TaqMan-MGB probe that is at a 5' end of the product of the first circulation, Tag DNA polymerase excises directional obstacles along a 5' to 3' direction by utilizing characteristics of 5' to 3' excision enzyme; the TaqMan-MGB probe further extends, and luminophore (R) at the 5' end of the TaqMan-MGB probe frees; the phenomenon of energy transfer disappears; the fluorescence emitted by (R) is detected, and fluorescence intensity is in direct proportion to the amount of PCR products. The TaqMan-MGB probe of the primer arm shortens the distance between two primers and promotes detection sensitivity.

Description

technical field [0001] The invention relates to a PCR hepatitis B virus quantitative detection method and a kit, in particular to a primer arm TaqMan-MGB probe PCR hepatitis B virus HBV DNA quantitative detection method and a kit. Background technique [0002] Chronic hepatitis B is the number one infectious disease in my country and one of the leading causes of death in China at present. There are about 120 million people in China carrying hepatitis B virus (HBV), among whom about 30 million are chronic hepatitis B patients. Nucleoside analog drugs are aimed at inhibiting HBV replication at the level of genes and molecular biology. The advent of nucleoside analog drug antiviral therapy is a breakthrough in the treatment of chronic hepatitis B. Since 1999, when the first drug of nucleoside analogs, lamivudine (Hepridin), was widely used clinically in China, drug resistance caused by mutation of hepatitis B virus (YMDD mutation) is a new clinical problem, and YMDD mutation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 陈勇
Owner 淮安市第四人民医院
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