Rockfish viral nerve necrosis virogene diagnostic kit and detecting method thereof

A technology for nerve necrosis and genetic diagnosis, which is applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve problems such as difficulty in popularization, troublesome material preparation, and low sensitivity, so as to improve scientific management efficiency and avoid virus transmission and epidemics , Guaranteed rapid effect

Inactive Publication Date: 2003-10-15
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some of these detection methods have high technical requirements, long detection time, troublesome preparation of materials required by some, high cost, and low sensitivity and low specificit

Method used

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  • Rockfish viral nerve necrosis virogene diagnostic kit and detecting method thereof

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Embodiment 1

[0024] Embodiment 1: Gene diagnostic kit of grouper viral neuronecrosis virus 1. RNA extraction liquid (A liquid), 2 tubes, 5ml / tube, Trizol liquid is installed inside. 2. Solution B, self-prepared, mainly chloroform, 200μl / part x 10 parts, 2ml in total. 3. Solution C, self-prepared, mainly isoamyl alcohol, 500μl / part x 10 parts, 5ml in total. 4. Liquid D, self-prepared, mainly 70% ethanol, 1ml / part x 10 parts, 10ml in total. 5. Liquid E, 1 tube, 0.5ml / tube, filled with DEPC water. 6. RT reaction solution (solution F), 1 tube, 0.1ml / tube, containing RT reaction solution (10μl system), including 5×Buffer, dNTP, DEPC-H 2 O, random primers, RNase inhibitor RNAsin (RI), reverse transcriptase M-MLV (RT). 7. RT-PCR reaction solution (solution G), 1 tube, 0.5ml / tube, containing RT-PCR reaction solution (50μl system), including 10×Buffer (including mg 2+ ), dNTP, forward primer, reverse primer, ddH 2 O and TaqE. 8. Positive control (solution H), 1 tube, 20ul / tube, containing VNN...

Embodiment 2

[0042] Embodiment 2: detection method of grouper viral neuronecrosis virus

[0043] Using the kit of Example 1, proceed as follows: 1. Take 0.1 g of the sample and add 1 ml of solution A to dilute, homogenize in an ice bath in a homogenizer, and stand at room temperature for 3 to 5 minutes. 2. Add 200 μl of solution B to the above homogenate, mix by inverting up and down, and let stand for 5 minutes. Centrifuge at 12000r / min for 15min at 3.4°C. 4. Take 400 μl supernatant, add it to 500 μl solution C, shake gently, and let stand for 10 minutes. 5.4°C, centrifuge at 12000r / min for 15min. 6. Discard the supernatant, wash twice with 1ml pre-cooled solution D, and centrifuge at 7500r / min at 4°C for 5min. 7. Air-dry or blow-dry on an ultra-clean workbench, add 50 μl E liquid water to dissolve (if it cannot be completely dissolved, it can be placed at 55-60°C for 10 minutes). The resulting RNA extract can be stored at -20°C. 8. Preheat the RNA extraction solution at 95°C for 5 m...

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Abstract

The present invention is rockfish viral nervous virus gene diagnosing kit and its usage. The kit and its usage is designed based on two pairs of primers for the conservation sequence of rockfish viral nervous virus gene. By means of PCR technology, the present invention detects qualitatively the specific DNA nucleic acid segment of tiger frog virus fast, simply, specifically and sensitively. The present invention may be used in the tracing detection of different stages of rockfish cultivation and environment monitoring.

Description

technical field [0001] The invention relates to a diagnostic kit and a detection method for diseases of aquatic economic animals, and mainly relates to a kit and a detection method for gene diagnosis of grouper viral nerve necrosis virus (VNNV, Viral Nervous Necrosis Virus). Background technique [0002] Grouper is one of the famous and valuable marine fishes in the world. Because of its fast growth and high and stable market price, it is very popular among farmers. It is a traditional marine fish in southern my country. In recent years, with the rapid development of my country's marine aquaculture industry, the scale of grouper culture in cages has also continued to expand. Due to the increase in the number of cages in the breeding sea area and the increase in the breeding density, the environment of the breeding waters is gradually deteriorating, and the occurrence of diseases is becoming more and more frequent. Common diseases include parasitic diseases, bacterial diseas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 何建国黄剑南黄志坚邓敏吕玲陈晓艳翁少萍
Owner SUN YAT SEN UNIV
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