Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof

A spleen and kidney necrosis virus and gene diagnosis technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of troublesome material preparation, difficulty in popularization, limited application and development, etc., to avoid virus spread and epidemic, The effect of improving scientific management efficiency and high practical value

Inactive Publication Date: 2003-10-15
SUN YAT SEN UNIV
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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, high cost, and low sensitivity and low specificity of some methods. Therefore, it is very difficult for the majority of farmers to master key technologies and it is difficult to promote , limiting the application and development of these technologies

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  • Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof

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Embodiment 1

[0027] Embodiment 1: the genetic diagnosis kit of mandarin fish infectious spleen and kidney necrosis virus

[0028] The kit consists of the following components (10 samples): 1. Release solution (solution A), 2 tubes, 5ml / tube, containing phosphate buffer (1×PBS), pH7.4. 2. Template extract ( Solution B), self-prepared or prepared, phenol / chloroform / isoamyl alcohol, the ratio is 25:24:1. 3. Solution C, 1 tube, 200μl / tube, filled with 5M NaCl. 4. Liquid D, self-prepared, 1ml / part×10parts, 10ml, mainly absolute ethanol. 5. E liquid, self-prepared, mainly 70% ethanol. 6. Solution F, 1 tube, 30μl / part x 10 parts, 300μl / tube, filled with sterilized double distilled water. 7. PCR reaction solution (solution G), 1 tube, 25μl / part × 10 copies, 250μl / tube, containing PCR first amplification reaction solution (25μl system), including ddH 2 O, 10×Buffer (including mg 2+ ), dNTP, outer primer F1, outer primer R1 and TaqE. 8. PCR reaction solution (solution H), 1 tube, 25μl / part × 10...

Embodiment 2

[0051] Embodiment 2: detection method of mandarin fish infectious spleen and kidney necrosis virus

[0052] Use the kit of embodiment 1, carry out according to the following steps: 1. sample 0.1g, add sample diluent (A solution) 1ml to dilute 10 times, homogenize in ice bath in homogenizer. 2. Centrifuge at 4000r / min for 10min. 3. Take 600 μl of supernatant / extract with template extract solution (solution B), invert up and down several times and mix well. 4. Centrifuge at 12000r / min for 10min. 5. Take 500ul supernatant and add 20μl / C solution, then add 1ml D solution, and precipitate at -20℃ for 1 hour. 6. Centrifuge at 12000r / min for 10min. 7. Discard the supernatant, add E solution to wash, centrifuge at 12000r / min for 5min, and wash twice. 8. Discard the supernatant and dry it naturally or on a clean bench. 9. Add 20-30 μl F solution to resuspend and dissolve as template. 10. Take 2 μl of the template and solution I respectively, add them to the PCR reaction solution ...

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Abstract

The present invention is mandarin fish infectious spleen and kidney necrosis 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 mandarin fish infectious spleen and kidney necrosis virus gene. By means of PCR technology, the present invention detects qualitatively the specific DNA nucleic acid segment of mandarin fish infectious spleen and kidney necrosis virus fast, simply, specifically and sensitively. The present invention may be used in the tracing detection of different stages of mandarin fish 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, in particular to a kit and a detection method for gene diagnosis of mandarin fish Infectious Spleen and Kidney Necrosis Virus (ISKNV, Infectious Spleen and Kidney Necrosis Virus). Background technique [0002] Mandarin fish is a famous freshwater cultured species in my country. With the success of mandarin fish artificial breeding and nursery technology, mandarin fish aquaculture has developed rapidly, and at the same time, disease problems have become increasingly serious. The diseases of mandarin fish mainly include viral diseases, bacterial diseases and parasitic diseases. Among them, viral diseases are prevalent rapidly and have a high incidence rate, and there are no effective control measures at present, which seriously affects the development of mandarin fish aquaculture. In recent years, large-scale explosive epidemics have occurred in f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 何建国邓敏翁少萍黄志坚吕玲
Owner SUN YAT SEN UNIV
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