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A fluorescent quantitative PCR kit for detecting bovine adenovirus type 3

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

Active Publication Date: 2012-02-01
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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  • A fluorescent quantitative PCR kit for detecting bovine adenovirus type 3
  • A fluorescent quantitative PCR kit for detecting bovine adenovirus type 3

Examples

Experimental program
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Effect test

Embodiment 1

[0029] The fluorescent quantitative PCR kit composition and reaction condition thereof of embodiment 1 bovine adenovirus type 3:

[0030] A). Kit composition:

[0031] a) The composition of the kit is as follows: (10 reactions)

[0032] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0033] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0034] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0035] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0036] Strong positive standard (100 times diluted PCR reaction tube (sterile, RNase and DNase free)

[0037] Release standard product 20μl / tube, four tubes in total) Nuclease H-free 2 O (2ml / tube)

[0038] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0039] (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

[0050] Example 2 Application of fluorescent quantitative PCR kit in the epidemiological investigation of adenovirus type 3 infection in dairy cows

[0051] A). Kit composition:

[0052] a) The composition of the kit is as follows: (10 reactions)

[0053] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0054] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0055] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0056] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0057] Strong positive standard (100 times diluted PCR reaction tube (sterile, RNase and DNase free)

[0058] Release standard product 20μl / tube, four tubes in total) Nuclease H-free 2 O (2ml / tube)

[0059] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0060] (DNA extraction solutions A, B, C, and D are products of TIANGEN Company. b) Hot-start Taq DNA polymerase (2U / μl), c)...

Embodiment 3

[0084] Example 3 Application of Fluorescent Quantitative PCR Kit in Quality Monitoring of Commercially Available Bovine Serum Products

[0085] A). Kit composition:

[0086] a) The composition of the kit is as follows: (10 reactions)

[0087] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0088] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0089] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0090] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0091] Strong positive standard (100 times diluted PCR reaction tube (sterile, RNase and DNase free)

[0092] Release standard product 20μl / tube, four tubes in total) Nuclease H-free 2 O (2ml / tube)

[0093] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0094] (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 fluorescent quantitative PCR kit for detecting bovine adenovirus type 3 and its application. The kit contains: a) a DNA extraction reagent, b) a hot-start Taq DNA polymerase, c) a primer and a TaqMan probe, d) ) Standard positive DNA template, e) PCR fluorescent quantitative reaction solution, characterized in that: the primer sequences are respectively sense primer: 5'-CCTGAATTCTCTTGCAGCCAGA-3', antisense primer: 5'-CCTACCGAACCGACGCAGAT-3', and the amplicon size is 100bp, the fluorescent probe sequence is: 5′-FAM-TGAGAAGGTACTCCTCGTCGCTGGACCA-TAMRA-3′, the 5′ end of the probe is labeled with the fluorescent emitting group FAM, and the 3′ end is labeled with the fluorescent quenching group TAMRA, and the standard positive DNA template is prepared by The pGEM-T vector inserted with the 100bp fragment of the adenovirus type 3 pol protein was transformed into Escherichia coli DH5α, and after multiplication, the plasmid was extracted and prepared, and quantified by measuring A260 with a UV spectrophotometer and 10-fold serial dilution. Efficient and convenient real-time monitoring of bovine adenovirus type 3 contamination in serum products can be widely used in epidemiological investigations of the virus infection, and can also provide technical support for related basic research, with broad application prospects.

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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Patent Type & Authority Patents(China)
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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