Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I

A real-time fluorescence quantitative, reovirus technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome operation, long cycle, unable to quantitative analysis, etc., to achieve detection sensitivity high effect

Inactive Publication Date: 2010-09-15
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods are either cumbersome to operate, have a long cycle, or

Method used

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  • Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I
  • Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Treat the extracted grass carp reovirus total RNA with lysate and RNA extractant;

[0035] 2. Primer design:

[0036] According to the grass carp hemorrhagic disease-873 outer capsid protein (VP7) gene sequence (AF403396) in GenBank, two pairs of specific primers were designed, and the length of the amplified fragment was 98bp. The designed primers were as follows:

[0037] Upstream primer: 5'-GGAATTCACCACGATGCCACTTCAC-3'

[0038] Downstream primer: 5'-CGGGATCCCGGTGCTTAATCGGATGG-3';

[0039] 3. RT-PCR amplification of grass carp reovirus outer capsid protein (VP7) gene:

[0040] Take 1 μg total virus RNA, add 20pmol upstream primer, follow Improm-Ⅱ TM ReverseTranscription System instructions for reverse transcription. Take 8 microliters of reverse transcription product cDNA, add 5 microliters of 10×Taq reaction buffer, 4 microliters of 2.5 mmol / L dNTPs, 50 micromol 1 / liter of dNTPs, 1 microliter of upstream and downstream, sterilized double distilled water 30.5...

Embodiment 2

[0051] 1. Treat the extracted grass carp reovirus total RNA with lysate and RNA extractant;

[0052] 2. Primer design:

[0053] According to the grass carp hemorrhagic disease-873 outer capsid protein (VP7) gene sequence (AF403396) in GenBank, two pairs of specific primers were designed, and the length of the amplified fragment was 98bp. The designed primers were as follows:

[0054] Upstream primer: 5'-GGAATTCACCACGATGCCACTTCAC-3'

[0055] Downstream primer: 5'-CGGGATCCCGGTGCTTAATCGGATGG-3';

[0056] 3. RT-PCR amplification of grass carp reovirus outer capsid protein (VP7) gene:

[0057] Take 5μg total virus RNA, add 20pmol upstream primer, follow Improm-Ⅱ TM ReverseTranscription System instructions for reverse transcription. Take 8 microliters of reverse transcription product cDNA, add 5 microliters of 10×Taq reaction buffer, 4 microliters of 2.5 mmol / L dNTPs, 50 micromol 1 / liter of dNTPs, 1 microliter of upstream and downstream, sterilized double distilled water 30.5 ...

Embodiment 3

[0068] 1. Treat the extracted grass carp reovirus total RNA with lysate and RNA extractant;

[0069] 2. Primer design:

[0070] According to the grass carp hemorrhagic disease-873 outer capsid protein (VP7) gene sequence (AF403396) in GenBank, two pairs of specific primers were designed, and the length of the amplified fragment was 98bp. The designed primers were as follows:

[0071] Upstream primer: 5'-GGAATTCACCACGATGCCACTTCAC-3'

[0072] Downstream primer: 5'-CGGGATCCCGGTGCTTAATCGGATGG-3';

[0073] 3. RT-PCR amplification of grass carp reovirus outer capsid protein (VP7) gene:

[0074] Take 10μg total virus RNA, add 20pmol upstream primer, follow Improm-Ⅱ TM ReverseTranscription System instructions for reverse transcription. Take 8 microliters of reverse transcription product cDNA, add 5 microliters of 10×Taq reaction buffer, 4 microliters of 2.5 mmol / L dNTPs, 50 micromol 1 / liter of dNTPs, 1 microliter of upstream and downstream, sterilized double distilled water 30.5 ...

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Abstract

The invention provides a real-time fluorescence quantitative PCR detection method of grass carp reovirus SYBR Green I, at least comprising the following steps of: extracting the total RNA of the grass carp reovirus, designing a primer, carrying out RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification on the coat protein gene of the grass carp reovirus, cloning and identifying the coat protein gene of the grass carp reovirus, diluting a standard template and establishing a fluorescence quantitative PCR standard curve. The detection method has low requirements on the concentration of the template and high detection sensitivity and can carry out quantitative analysis. The detection method can detect 72 samples at a time; a result can be observed immediately once the PCR reaction is finished without the operations of electrophoresis, dyeing and the like, and therefore, the detection method is more convenient and rapid than conventional RT-PCR methods.

Description

technical field [0001] The invention relates to the technical field of in vitro molecular diagnosis of biomedicine, in particular to a real-time fluorescent quantitative PCR detection method for grass carp reovirus SYBR GreenI. Background technique [0002] Grass carp hemorrhagic disease (Hemorrhaged disease of grasscarp) is a serious infectious disease infecting grass carp. The epidemic season is long, and the morbidity and mortality are high, often resulting in the death of a large number of grass carp fry; 1-year-old herring is also affected, and grass carp over 2 years old sometimes suffer from this disease. The pathogen of grass carp haemorrhagic disease is Grass Carp Reovirμs (GCRV), which is the most virulent virus among the viruses of the family Reoviridae and the genus Aquatic Reovirus. Grass carp reovirus genome consists of 11 segmented dsRNAs with a total molecular weight of 15.46×10 6 Dalton encoded 12 polypeptides, and studies have shown that the relationship ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 周勇曾令兵范玉顶罗晓松徐进马杰
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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