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Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting mouse adenoviruses and application

A fluorescence quantification and mouse adenovirus technology, which is applied in the field of biotechnology related to the invention, can solve problems such as the inability to quantify DNA or RNA copy numbers, and achieve the effects of reducing experimental costs, accurate quantification, and broad application prospects.

Active Publication Date: 2013-07-24
CANVEST WUHAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is no linear relationship between the amount of end product of PCR and the amount of starting template, it is not possible to quantify the starting DNA or RNA copy number

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] A fluorescent quantitative PCR kit for detecting mouse adenovirus, the kit composition and its reaction conditions are:

[0034] A) The composition of the kit is as follows: (10 reactions, 10 samples to be tested)

[0035] DNA extraction solution (10mL / tube), strong positive standard (50μL / tube), PCR reaction tube (sterile)

[0036] Negative standard (50 μL / tube), borderline positive standard (50 μL / tube);

[0037] Strong positive standard, i.e. copy number 10 8 Standard positive DNA template for c / r;

[0038] Negative standard, that is, DNA from L929 cells (host cells of mouse adenovirus) not infected with mouse adenovirus;

[0039] Borderline positive standard, i.e. copy number 10 2 Standard positive DNA template for c / r.

[0040] SYBR Green I real-time fluorescent quantitative PCR reaction solution (110 μL / tube) (d) SYBR Green I real-time fluorescent quantitative PCR reaction solution, purchased from Bio-rad company)

[0041] The DNA extraction solution (50mL) ...

Embodiment 2

[0055] The application of a mouse adenovirus fluorescent quantitative PCR kit in the detection of L929 cell infection mouse adenovirus drug research, the application process is:

[0056] A) The composition of the kit is as follows: (10 reactions)

[0057] DNA extraction solution (10mL / tube);

[0058] Strong positive standard (50μL / tube);

[0059] PCR reaction tube (sterile);

[0060] Negative standard (50μL / tube);

[0061] Borderline positive standard (50 μL / tube);

[0062] SYBR Green I real-time fluorescent quantitative PCR reaction solution (110 μL / tube) (d) SYBR Green I real-time fluorescent quantitative PCR reaction solution, purchased from Bio-rad Company).

[0063] B) Virus extraction process from L929 cells: culture L929 cells infected or not infected with mouse adenovirus in T25 cell culture flasks, add 500 μL of a) DNA extraction solution, digest at 37°C for 3 hours; add to the cell digestion solution Equal volume of isopropanol, mix gently, and centrifuge at 10,...

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Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting mouse adenoviruses and an application. The kit comprises (a) a DNA (Deoxyribonucleic Acid) extracted solution, (b) a primer, (c) a standard positive template and (d) a real-time SYBR Green I fluorescent quantitative PCR reaction solution, and is characterized in that DNAs are extracted from a cell sample to carry out fluorescent quantitative PCR reaction; meanwhile, the standard positive template is led in; primer sequences comprise a positive primer: 5'-ATAAGAAAGGATGCGGAAAAGGAC-3', and a negative primer: 5'-CCCAAAACAGAAGCAACAGAGTAA-3'; the amplicon is 170bp; the standard positive DNA template is connected with a pTA2 carrier of a protein encoding region 170bp through a conservative virus DNA inserted into the mouse adenoviruses to transform bacillus coli DH5alpha; and proliferation is carried out and then plasmids are extracted, and an ultraviolet spectrophotometer measures A260 to quantify and 10 times of gradient dilution is carried out. The result is accurate and reliable; the stability is good; and the repeatability is good. The sensitivity is high, the quantification is rapid and accurate and the detection range is wide. The fluorescent quantitative PCR kit can carry out epidemiological survey caused by the mouse adenoviruses and can provide technical support for a relative basic research; and an application prospect is very wide.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to a SYBR Green I real-time fluorescent quantitative PCR kit for detecting mouse adenovirus, and also to the application of the SYBR Green I real-time fluorescent quantitative PCR kit. The SYBR Green I real-time fluorescent quantitative PCR kit The PCR kit can efficiently and conveniently carry out quantitative detection and quality control of mouse-derived adenoviruses in various biological materials. It can also conduct epidemiological investigations of mouse adenoviruses, and can also provide technical support for related basic research. The application prospect is very promising widely. Background technique [0002] Murine adenovirus 1 (MAdV-1) belongs to the genus Mastad enovirus of the family Adenoviridae. Adenovirus has no envelope, and the virion is icosahedral, with a diameter of 80-110nm, consisting of 252 capsomers. The relative molecular mass of adenoviruses generally ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 肖庚富张哲郑从义
Owner CANVEST WUHAN BIOTECH