Macrobrachium rosenbergii nodavirus detection kit and detection method

A Nodamura virus and detection kit technology, which is applied to the detection kit and detection field of Macrobrachium rosenbergii Nodamura virus, can solve the problems of inability to detect virus-carrying shrimp, unsatisfactory sensitivity, complicated operation, etc., and achieve programmability and standardization, the detection results are intuitive and clear, and the detection specificity is high

Inactive Publication Date: 2013-05-29
杭州三合创新科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although electron microscopy can directly observe virus particles, the operation is complex, time-consuming, and low in accuracy; although the antibody detection method for the detection of Macrobrachium rosenbergii Nodamura virus has been recommended by OIE, its sensitivity is not ideal, and it is usually unable to detect no symptoms Potential virus-carrying shrimp; Macrobrachium rosenbergii Nodamura virus RT-PCR detection method has good sensitivity, but it has high requirements for laboratories and operators, and it cannot achieve relatively fast and accurate detection of Macrobrachium rosenbergii Nodamura virus. It is even more impossible to realize on-site rapid detection of viruses, which greatly limits the popularization and application of RT-PCR detection methods in production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Macrobrachium rosenbergii Nodamura virus detection kit, which includes the amplification reaction solution containing primers; the components are: the upstream primer F1 and the downstream primer R1 of the amplification primer are each 1~2uM, and the upstream primer F2 and the downstream primer R2 of the amplification primer are Each 0.1~0.4uM, detection probe F3 and detection probe R3 of amplification primers 0.1~0.4uM each, dATP, dTTP, dGTP, dCTP each 0.8~2.0mM, MgCl2 4~10 mM, betaine 0.6~1.2M , Tris-HCL 10~40 mM, KCl 10~20 mM, MgSO 4 1~4 mM, (NH 4 ) 2 SO 4 6~12 mM, Triton X-100 0.5%~1.0%, reverse transcriptase 1~20u, DNA polymerase with strand displacement activity 2~20u.

[0044] Primers are preferably as follows:

[0045] Upstream primer F1: 5'-tagatatgttcctgcagtacc-3' SEQ ID NO: 11

[0046] Downstream primer R1: 5'-tcacttgcaagaggtaaaat-3' SEQ ID NO:12

[0047] Upstream primer F2:

[0048] 5'-acttgtgacgtagcctgcctttttgatacaaatccactagatgacc-3' SEQ ID NO: 13 ...

Embodiment 2

[0070] A kit for detecting Macrobrachium rosenbergii Nodamura virus, comprising:

[0071] Sample lysate; (The solution configuration method is: dissolve 60~240g GuSCN in 50~200mL 0.1mol / L Tris-HCl (pH6.4), add 10~40mL 0.2mol / L EDTA, pH8.0 and 2~6mL Just mix TritonX-100)

[0072] Proteinase K; (prepare a solution containing 50mmol / L TRIS-CL (PH8.0), 1.5mmol / L calcium acetate and 50% glycerol, after autoclaving, dissolve proteinase K powder in the mixed solution to a concentration of 20mg / ml, Store the prepared proteinase K solution at -20°C.)

[0073] Sample cleaning solution; (solution composition: 50~85% ethanol and sodium acetate with a final concentration of 3~300mmol / L)

[0074] Nucleic acid eluent; (20mmol / L TE solution)

[0075] Sample enrichment column; (purchased from the market)

[0076] Constant temperature amplification reaction solution; (the components are as follows): 1~2uM each of the upstream primer F1 and downstream primer R1 of the amplification primer, 0...

Embodiment 3

[0097] According to Embodiments 1 and 2, this embodiment specifically discloses a usage method, and due to limited space, the formula, sequence, etc. will not be repeated here.

[0098] 1. Sample processing: Take 50-100mg of the tissue to be tested (fresh or -70°C or stored in liquid nitrogen) into a 1.5ml centrifuge tube, add 1ml Trizol to fully homogenate, and let stand at room temperature for 5 minutes.

[0099] 2. Add 0.2ml of chloroform, shake for 15s, and let stand for 2min.

[0100] 3. Centrifuge at 4°C, 12000g×15min, and take the supernatant.

[0101] 4. Add 0.5ml of isopropanol, mix the liquid in the tube gently, and let stand at room temperature for 10min.

[0102] 5. Centrifuge at 4°C, 12000g×10min, discard the supernatant.

[0103] 6. Add 1ml of 75% ethanol and gently wash the pellet. 4°C, 7500g×5min, discard the supernatant.

[0104] 7. Dry it in the air, add an appropriate amount of DEPC H2O to dissolve (65°C for 10-15 minutes).

[0105] 8. Constant ...

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Abstract

The invention discloses a macrobrachium rosenbergii nodavirus detection kit and detection method. According to the detection kit, with the combination of the characteristics of isothermal amplification and rapid detection of nucleic acid test strips, expensive instrument equipment is not required during the detection process; and the color display is performed by utilizing the nucleic acid test strip, so that the detection reliability and the result judgment easiness are improved. The detection kit is low in detection cost and convenient to use and is safe for people and environment, and is capable of replacing the existing relevant detection methods. The detection kit can be used on wild production site and in laboratories in various levels, and has popularization and application value in the aspects of enhancing macrobrachium rosenbergii nodavirus detection, discovering and controlling the individual propagation of infected macrobrachium superbum infected by virus in real time, discovering and preventing the large-scale epidemic outbreak of the epidemix disease and the like.

Description

technical field [0001] The invention relates to a detection kit and a detection method for detecting related viruses in aquaculture, in particular to a detection kit and a detection method for detecting Macrobrachium rosenbergii Nodamura virus. Background technique [0002] Macrobrachium rosenbergii is also known as Malaysian prawn, money prawn, etc. It belongs to the order Decapodidae, Longarm Prawnidae, Macrobrachium genus in classification, and is known as the king of freshwater macrobrachium. Native to the Indo-Pacific region along the coast of Ecuador, it is currently one of the three most widely farmed shrimp species in the world. By 2010, the global production of Macrobrachium rosenbergii had reached more than 200,000 tons, making it the freshwater shrimp with the highest aquaculture output in the world. Among them, my country's aquaculture output reached more than 100,000 tons, making it the world's largest macrobrachium rosenbergii farming country. [0003] Around ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 石坚钱东林峰宋之琦徐锦余
Owner 杭州三合创新科技有限公司
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