Bovine parvovirus detecting fluorescence quantitative PCR kit and application thereof

A parvovirus, fluorescence quantitative technology, applied in fluorescence/phosphorescence, microbe-based methods, microbe assay/inspection, etc., can solve the problem of inability to calculate the initial DNA copy number

Active Publication Date: 2009-10-28
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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  • Bovine parvovirus detecting fluorescence quantitative PCR kit and application thereof
  • Bovine parvovirus detecting fluorescence quantitative PCR kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Composition and reaction conditions of a fluorescent quantitative PCR kit for bovine parvovirus:

[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 dilute PCR reaction tube (sterile, no RNase and DNase)

[0037] Interpretation value standard product 20μl / tube, a total of four tubes) Nuclease-free H 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 amplification reaction solut...

Embodiment 2

[0051] Example 2 Application of Fluorescent Quantitative PCR Kit in Epidemiological Investigation of Parvovirus Infection in Dairy Cows

[0052] A). Kit composition:

[0053] a) The composition of the kit is as follows: (10 reactions)

[0054] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0055] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0056] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0057] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0058] Strong positive standard (100 times diluted PCR reaction tube (sterile, no RNase and DNase)

[0059] Interpretation value standard product 20μl / tube, a total of four tubes) Nuclease-free H 2 O(2ml / tube)

[0060] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0061] (DNA extraction solutions A, B, C, and D are products of TIANGEN. b) Hot-start Taq DNA polymerase (2U / μl), c) PCR reaction solu...

Embodiment 3

[0085] Example 3 Application of Fluorescence Quantitative PCR Kit in Quality Monitoring of Commercial Bovine Serum Products

[0086] A). Kit composition:

[0087] a) The composition of the kit is as follows: (10 reactions)

[0088] DNA extraction solution A (4ml / tube) DNA extraction solution B (2ml / tube)

[0089] DNA extraction solution C (2.6ml / tube) DNA extraction solution D (2.5ml / tube)

[0090] RNaseA (40ul / tube) Proteinase K solution (2ml / tube)

[0091] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0092] Strong positive standard (100 times diluted PCR reaction tube (sterile, no RNase and DNase)

[0093] Interpretation value standard product 20μl / tube, a total of four tubes) Nuclease-free H 2 O(2ml / tube)

[0094] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0095] (DNA extraction solutions A, B, C, and D are products of TIANGEN. b) Hot-start Taq DNA polymerase (2U / μl), c) PCR reaction solution purchas...

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Abstract

The invention discloses a bovine parvovirus detecting fluorescence quantitative PCR kit and an application thereof. The kit comprises: a) a DNA extraction reagent, b) a hot start Taq DNA polymerase, c) a primer and a TaqMan probe, d) a standard positive DNA template, and e) a PCR fluorescence quantitative reaction solution. The kit is characterized in that: primer sequence is a sense primer: 5'-CCAGTACCAGGAAACGGAGAC-3', and an antisense primer: 5'-GCATGTATTCCGGTCTCCAA -3', and the size of an amplicon is 118 bp; the sequence of a fluorescence probe is: 5'-FAM-CCTCAACATCTACGTCACCGGACAA-TAMRA-3', the 5' end of the probe is marked with a fluorescence emission group FAM, the 3' end is marked with a fluorescence quenching group TAMRA, the standard positive DNA template transforms a colon bacillus DH5 alpha by a pGEM-T carrier inserted with bovine parvovirus VP3 protein coding zone 118 bp segment, plasmid is extracted after proliferation, an A260 ration is measured in an ultraviolet spectrophotometer, and the plasmid is diluted by 10 times of gradient. The fluorescence quantitative PCR kit, applied to the epidemiology investigation of cow bovine parvovirus infection, can efficiently and conveniently monitor the bovine parvovirus pollution in serum products in real time, and is widely applicable to the epidemiology investigation of bovine parvovirus infection.

Description

Technical field [0001] The present invention relates to the field of biotechnology, relates to a fluorescent quantitative PCR kit for detecting bovine parvovirus, and also relates to the use of the fluorescent quantitative PCR kit. It is suitable for real-time monitoring of bovine parvovirus contamination in serum products and is widely used at the same time. For the epidemiological investigation of the virus infection, it can also provide technical support for related basic research. Background technique [0002] Bovine parvovirus (BPV) belongs to the Parvovirus genus of the Parvoviridae family. Its genome is a single-stranded DNA molecule, which mainly causes respiratory and intestinal diseases in cattle. Its main feature is to cause bovine diarrhea and cause abortion in cows. Artificial oral or intravenous infection of newborn calves without colostrum and no antibodies in their bodies will develop diarrhea within 24 to 48 hours, accompanied by viremia. In addition, the incubat...

Claims

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Application Information

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