Bovine parainfluenza type-3 virus detecting fluorescence quantitative PCR kit and application thereof

A type 3 virus, 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 or RNA 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 or RNA copy number cannot be calculated based on the amount of final PCR product

Method used

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  • Bovine parainfluenza type-3 virus detecting fluorescence quantitative PCR kit and application thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0030] The fluorescent quantitative PCR kit composition and reaction condition thereof of embodiment 1 bovine parainfluenza type 3 virus

[0031] A). Kit composition:

[0032] a) The composition of the kit is as follows: (10 reactions)

[0033] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0034] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0035] RNase inhibitor (400U / tube)

[0036] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0037] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

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

[0039] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0040] (RNA extraction solution A is a product of Invitrogen Company. b) reverse transcription reaction solution, d) reverse transcriptase (purchased from Promega Company, g)...

Embodiment 2

[0060] Example 2: Application of Bovine Parainfluenza Type 3 Virus Fluorescent Quantitative PCR Kit in the Epidemiological Investigation of Cows Infected with Bovine Parainfluenza Type 3 Virus

[0061] A). Kit composition:

[0062] a) The composition of the kit is as follows: (10 reactions)

[0063] RNA extraction solution A (5ml / tube) RNA extraction solution B (5ml / tube)

[0064] RNA extraction solution C (10ml / tube)

[0065] Filter column A and adsorption column B (sterile, RNase and DNase free)

[0066] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0067] RNase inhibitor (400U / tube)

[0068] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0069] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0070] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0071] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl...

Embodiment 3

[0091] Example 3: Application of Bovine Parainfluenza Type 3 Fluorescent Quantitative PCR Kit in Detection of Commercially Available Bovine Serum

[0092] A). Kit composition:

[0093] a) The composition of the kit is as follows: (10 reactions)

[0094] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0095] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0096] RNase inhibitor (400U / tube)

[0097] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0098] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0099] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0100] PCR amplification reaction solution (220μl / tube) Hot start Taq DNA polymerase (2.75μl / tube)

[0101] (RNA extraction solution A is a product of Invitrogen Company. b) reverse transcription reaction solution, d) reverse transcriptase was purchased from Promega...

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Abstract

The invention discloses a bovine parainfluenza type-3 virus detecting fluorescence quantitative PCR kit and an application thereof. The kit comprises: a) an RNA extraction solution, b) a reverse transcriptase reaction solution, c) reverse transcriptase, d) an RNA enzyme inhibitor, e) a primer and a TaqMan probe, f) a standard positive DNA template, and g) a fluorescence quantitative PCR reaction solution. The kit is characterized in that primer sequence is respectively a sense primer and an antisense primer: the size of an amplicon is 129 bp; in the sequence of a fluorescence probe, the 5' end of the probe is marked with a fluorescence emission group FAM while 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 parainfluenza type-3 virus conservative F protein coding zone 188 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 result of the kit is more accurate, reliable, stable and repeatable. The kit can also be applied to the epidemiology investigation of bovine epidemic influenza and provide technical support for relevant fundamental researches, thus having quite broad application prospect.

Description

Technical field: [0001] The invention relates to the field of biotechnology, more specifically to a fluorescent quantitative PCR kit for detecting bovine parainfluenza type 3 virus, and also relates to the use of the fluorescent quantitative PCR kit, which can efficiently and conveniently detect Quantitative detection and quality control of bovine blood products and bovine serum can also be used for epidemiological investigation of bovine parainfluenza, and can also provide technical support for related basic research, with a wide application prospect. Background technique [0002] Bovine parainfluenza virus type 3 (PI-3) belongs to the genus Respirovirus of the family Paramyxoviridae, and is a segmented negative-strand RNA virus with a diameter of virions of 120 ~ 180nm, nucleocapsid helical symmetry, containing hemagglutinin and neuraminidase activity. Serological studies have shown that the presence of PI-3 virus antibodies in healthy bovine serum is more common, so it i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 郑从义郭佳黄璇张国荣
Owner WUHAN SANLI BIO TECH
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