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Detection kit and detection method for grass carp hemorrhage virus

A technology of detection kits and detection methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The effect of improved detection efficiency, high sensitivity and specificity

Inactive Publication Date: 2018-08-17
转导精进(武汉)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Grass carp hemorrhagic virus is an RNA virus. The ordinary detection process is complicated, and the RNA is easily degraded, resulting in false negatives.

Method used

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  • Detection kit and detection method for grass carp hemorrhage virus
  • Detection kit and detection method for grass carp hemorrhage virus
  • Detection kit and detection method for grass carp hemorrhage virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Primer Design and Screening for Fluorescent Quantitative RT-PCR Detection of Grass Carp Hemorrhagic Virus VP5 Gene

[0049] Download the VP5 sequence of Grass Carp Hemorrhagic Virus (GeneBank: AF239175.1) collected by GenBank, and also download the genome of the negative control virus, killifish reovirus, spring dart perch reovirus, California perch reovirus, Malaysia For salmon reovirus, see Table 1 below. After comparison by Clustal X, select the appropriate region to design primers. This region is specifically expressed in grass carp hemorrhagic virus, but there is at least 10%-30% difference compared with the negative control. After the region is selected, use ABI Primer Express 3.0 in real time Fluorescent quantitative RT-PCR primer design software, designed synthetic primers. The extracted alternative primers were screened according to the following requirements:

[0050] 1) The primers should be designed in the conserved region of the nucleic acid ser...

Embodiment 2

[0122] The optimization of embodiment 2 fluorescence quantitative RT-PCR primer consumption

[0123] 1. The first optimization of the amount of fluorescent quantitative RT-PCR primers

[0124] With diluted L1 (10 -4 ) and L2 (10 -5 ) The positive control RNA was used as a template for optimizing the amount of primers, and the upstream and downstream amounts of the primers were gradient-optimized respectively. The concentration of the primer working solution was 10 pmol / μL. 2.5 μL of U / μL Taq DNA polymerase, 0.5 μL of 5 U / μL reverse transcriptase (QuantReverse Transcriptase), 25 μL of 2× imported real-time fluorescent PCR buffer (5 μL of 10× buffer purchased from ThermoFisher, 4 μL of 10 mM dNTP, 0.005 μL of 10000× SYBR green I and 16 μL DEPC water), add sterile water to 50 μL. The PCR reaction conditions were: 50°C for 30 min; 95°C for 3 min; 95°C for 10 sec, 60°C for 20 sec, 72°C for 20 sec, 75°C for 5 sec, and a total of 45 cycles for the read plate. The results are show...

Embodiment 3

[0137] Example 3 Grass carp hemorrhagic virus VP5 gene fluorescence quantitative RT-PCR detection

[0138] 1. Establishment of standard curve

[0139] The positive control RNA was used as a template for fluorescent quantitative RT-PCR detection to establish a standard curve. The specific operation is as follows: the positive control RNA was serially diluted 10 times to I: 1.0×10 5 pg / μL; II: 1.0×10 4 pg / μL; III: 1.0×10 3 pg / μL; IV: 1.0×10 2pg / μL; V: 10 pg / μL. The standard product detection PCR reaction system is: 3.0 μL of 10 pmol / μL upstream primer, 2.0 μL of 10 pmol / μL downstream primer, 5 μL of positive control RNA, 2.5 μL of 2.5 U / μL Taq DNA polymerase, 5 U / μL reverse transcriptase ( Quant Reverse Transcriptase) 0.5 μL, 12 μL DEPC water, 2× imported real-time fluorescent PCR buffer 25 μL (purchased from ThermoFisher 5 μL 10× buffer, 4 μL 10 mM dNTP, 0.005 μL 10000× SYBR green I and 16 μL DEPC water). The PCR reaction conditions were: 50°C for 30 min; 95°C for 3 min; ...

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Abstract

The present invention discloses a primer pair for grass carp hemorrhage virus VP5 gene sequence amplification, a grass carp hemorrhage virus VP5 gene sequence amplification method, a grass carp hemorrhage virus detection kit and a grass carp hemorrhage virus detection method. According to the present invention, compared to the reported method, the established one-step fluorescence quantitative RT-PCR method of the present invention has advantages of simpleness, rapidlyness, accuracy, sensitivity and low detection limit, and can quickly and accurately determine the virus and the content in thediseased fish body tissue.

Description

technical field [0001] The invention relates to the detection field of fish pathogenic virus, relates to a detection kit and detection method of fish pathogenic virus, in particular to a detection kit and detection method of grass carp hemorrhagic virus. Background technique [0002] Grass carp hemorrhage virus (GCHV, referred to as reovirus of grass carp, GCRV by the International Committee on Taxonomy of Viruses) is the first fish virus isolated in China, belonging to the family Reoviridae and the genus Reovirus of aquatic animals , 70-80nm in diameter, 20-hedron spherical particles, containing 11 segments of double-stranded RNA. Different strains exist in different regions. At present, 10 isolates have been reported. The virus mainly causes haemorrhagic disease in the fingerling stage of grass carp, the main freshwater aquaculture species in China, with a mortality rate of over 90%, causing huge losses to the aquaculture industry. [0003] Grass carp viral hemorrhage of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2547/101C12Q2563/107C12Q2545/113
Inventor 张金朱馨蕾
Owner 转导精进(武汉)生物技术有限公司
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