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Fluorescence quantitative PCR kit for detecting type-3 cow adenovirus and application

A fluorescence quantification and kit technology, applied in the direction of fluorescence/phosphorescence, microbe-based methods, microbiological measurement/inspection, etc., can solve the problem that the initial DNA copy number cannot be calculated, etc.

Active Publication Date: 2009-10-21
WUHAN SANLI BIO TECH
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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 final PCR product and the amount of starting template, the starting DNA copy number cannot be calculated based on the amount of final PCR product

Method used

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  • Fluorescence quantitative PCR kit for detecting type-3 cow adenovirus and application
  • Fluorescence quantitative PCR kit for detecting type-3 cow adenovirus and application
  • Fluorescence quantitative PCR kit for detecting type-3 cow adenovirus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The fluorescent quantitative PCR kit composition and reaction condition thereof of embodiment 1 bovine adenovirus type 3:

[0033] A). Kit composition:

[0034] a) The composition of the kit is as follows: (10 reactions)

[0035] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0036] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0037] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0038] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0039] Strong positive standard (100 times diluted PCR reaction tube (sterile, RNase and DNase free)

[0040] Release standard product 20μl / tube, four tubes in total) Nuclease H-free 2 O (2ml / tube)

[0041] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0042] (DNA extraction solutions A, B, C, and D are products of TIANGEN Company. b) Hot-start Taq DNA polymerase (2U / μl), c) PCR reaction solu...

Embodiment 2

[0053] Example 2 Application of fluorescent quantitative PCR kit in the epidemiological investigation of adenovirus type 3 infection in dairy cows

[0054] A). Kit composition:

[0055] a) The composition of the kit is as follows: (10 reactions)

[0056] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0057] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0058] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0059] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0060] Strong positive standard (100 times diluted PCR reaction tube (sterile, RNase and DNase free)

[0061] Release standard product 20μl / tube, four tubes in total) Nuclease H-free 2 O (2ml / tube)

[0062] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0063] (DNA extraction solutions A, B, C, and D are products of TIANGEN Company. b) Hot-start Taq DNA polymerase (2U / μl), c)...

Embodiment 3

[0087] Example 3 Application of Fluorescent Quantitative PCR Kit in Quality Monitoring of Commercially Available Bovine Serum Products

[0088] A). Kit composition:

[0089] a) The composition of the kit is as follows: (10 reactions)

[0090] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0091] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0092] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0093] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0094] Strong positive standard (100 times diluted PCR reaction tube (sterile, RNase and DNase free)

[0095] Release standard product 20μl / tube, four tubes in total) Nuclease H-free 2 O (2ml / tube)

[0096] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0097] (DNA extraction solutions A, B, C, and D are products of TIANGEN Company. b) Hot-start Taq DNA polymerase (2U / μl), c) PCR reactio...

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Abstract

The invention discloses a fluorescence quantitative PCR kit for detecting type-3 cow adenovirus and application. The kit comprises a) DNA extraction reagent, b) hot starting Taq DNA polymerase, c) primers and TaqMan probe, d) standard positive DNA template, and e) PCR fluorescence quantitative reaction liquid. The kit is characterized in that the sequence of a positive primer is 5'-CCTGAATTCTCTTGCAGCCAGA-3', the sequence of a negative primer is 5'-CCTACCGAACCGACGCAGAT-3', the size of an amplicon is 100bp, the sequence of a fluorescence probe is 5'-FAM-TGAGAAGGTACTCCTCGTCGCTGGACCA-TAMRA-3', a 5' end of the probe marks a fluorescence emitting group FAM, a near 3' end of the probe marks a fluorescence quenching group TAMRA, the standard positive DNA template converts colon bacillus DH5a by a pGEM-T carrier inserted into a type-3 adenovirus pol protein 100bp fragment, plasmids are extracted after multiplication to prepare the kit, and A260 is measured by an ultraviolet spectrophotometer to definite quantity and is diluted by 10 times of gradient. The kit efficiently and conveniently monitors type-3 cow adenovirus pollution in a serum product in real time, can be widely applied to epidemiology research on adenovirus infection, can provide technical support for related basic research, and has wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, relates to a fluorescent quantitative PCR kit for detecting bovine adenovirus type 3, and also relates to the use of the fluorescent quantitative PCR kit, which is suitable for real-time monitoring of bovine adenovirus type 3 pollution in serum products At the same time, it is widely used in the epidemiological investigation of the virus infection, and can also provide technical support for related basic research. Background technique [0002] Bovine adenovirus type 3 belongs to the Adenoviridae family of adenoviruses, and its genome is a double-stranded DNA molecule, which mainly causes respiratory and intestinal diseases in cattle. Its main features are pneumonia, enteritis, conjunctivitis and polyarthritis, and it is one of the important causes of calf death. Diseased and latently infected cattle can detoxify and can infect susceptible cattle in contact with it. Its susceptibility and latentness ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 郑从义郭佳张国荣
Owner WUHAN SANLI BIO TECH
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