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Reovirus-detecting fluorescence quantitative PCR kit and application thereof

A reovirus, fluorescence quantitative technology, applied in the direction of fluorescence/phosphorescence, microbe-based methods, microbiological measurement/inspection, etc., can solve the problem that the original DNA or RNA copy number cannot be calculated, etc.

Active Publication Date: 2011-08-17
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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  • Reovirus-detecting fluorescence quantitative PCR kit and application thereof
  • Reovirus-detecting fluorescence quantitative PCR kit and application thereof

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Effect test

Embodiment 1

[0029] The fluorescent quantitative PCR kit composition and reaction condition thereof of embodiment 1 reovirus

[0030] A). Kit composition:

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

[0032] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0033] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0034] RNase inhibitor (400U / tube)

[0035] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0036] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0037] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0038] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0039] (RNA extraction solution A is a product of Invitrogen Company. b) reverse transcription reaction solution, d) reverse transcriptase was purchased from Promega Company, g) fluorescent quantitative PCR reaction solution and RN...

Embodiment 2

[0059] Example 2: Application of Reovirus Fluorescent Quantitative PCR Kit in Epidemiological Investigation of Cows Infected with Reovirus.

[0060] a) The composition of the kit is as follows: (10 reactions)

[0061] RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0062] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0063] RNase inhibitor (400U / tube)

[0064] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0065] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0066] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0067] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0068] (RNA extraction solution A is a product of Invitrogen Company. b) reverse transcription reaction solution, d) reverse transcriptase was purchased from Promega Company, g) fluorescent quantitative PCR reaction solution and RNase...

Embodiment 3

[0098] Example 3: Application of Reovirus Fluorescent Quantitative PCR Kit in Detection of Commercially Available Bovine Serum

[0099] A). Kit composition:

[0100] a) The composition of the kit is as follows: (10 reactions)

[0101]RNA extraction solution A (4ml / tube) RNA extraction solution B (800μl / tube)

[0102] Reverse transcription reaction solution (90μl / tube) Reverse transcriptase (10μl / tube)

[0103] RNase inhibitor (400U / tube)

[0104] Strong Positive Standard (100-fold diluted standard 20μl / tube, four tubes in total)

[0105] PCR reaction tube (sterile, RNase and DNase free) DEPC H 2 O (2ml / tube)

[0106] Negative standard (50μl / tube) Borderline positive standard (50μl / tube)

[0107] Fluorescent quantitative PCR reaction solution (125μl / tube)

[0108] (RNA extraction solution A is a product of Invitrogen Company. b) reverse transcription reaction solution, d) reverse transcriptase was purchased from Promega Company, g) fluorescent quantitative PCR reaction s...

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Abstract

The invention discloses a reovirus-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 PCR fluorescence quantitative reaction solution. The kit is characterized in that: primer sequence is a sense primer: 5'-TGCGCCTATCCTTGAGTTGA-3', and an antisense primer: 5'-TTGCCAGGAAATACGGGTCT-3', and the size of an amplicon is 138 bp; the sequence of a fluorescence probe is: 5'-FAM-TCAAAATGGTGGACTTCAGTTTCGATTT-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, thestandard positive DNA template transforms a colon bacillus DH5 alpha by a pGEM-T carrier inserted with reovirus S1 protein zone 361 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 kit can efficiently and conveniently monitor the reovirus pollution in serum products in realtime, can be applicable to epidemiology investigation of reovirus infection, and can 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 reovirus, and also relates to the use of the fluorescent quantitative PCR kit, which is suitable for real-time monitoring of reovirus contamination 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] Reovirus belongs to the Reoviridae family (Reoviridae), with a segmented double-stranded RNA genome, no envelope, and a double-layer capsid. It has a wide host range and can infect humans, vertebrates, insects, and plants. 5 genera. It mainly causes diarrhea, dehydration and death in young animals in cattle herds, and is a common disease in dairy and beef cattle herds. The detection rate of antibodies in the herd is extremely high, and the mortality rate is very high during a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/92
Inventor 郑从义郭佳张国荣
Owner WUHAN SANLI BIO TECH
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