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CyHV-2 (cyprinid herpesvirus 2) specificity PCR (polymerase chain reaction) detection kit and detection method

A carp herpes virus and detection kit technology is applied in the field of aquaculture to achieve the effects of accurate detection, high detection sensitivity and rapid detection

Active Publication Date: 2015-08-19
广州利洋水产科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The diagnosis of traditional viral diseases requires methods such as virus isolation, cell culture, electron microscope observation, and immune detection, and there is a lack of rapid, accurate, and sensitive detection methods for viruses

Method used

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  • CyHV-2 (cyprinid herpesvirus 2) specificity PCR (polymerase chain reaction) detection kit and detection method
  • CyHV-2 (cyprinid herpesvirus 2) specificity PCR (polymerase chain reaction) detection kit and detection method
  • CyHV-2 (cyprinid herpesvirus 2) specificity PCR (polymerase chain reaction) detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Carp Carp Herpesvirus Type 2 PCR Rapid Detection Kit

[0046] All the chemical reagents and primers of the crucian carp herpesvirus type 2 PCR rapid detection kit described in this example were purchased from professional reagent companies. However, the sources of the above reagents and primers do not constitute any limitation to the present invention, and the present invention can prepare relevant reagents and synthesize relevant primers by itself.

[0047] The kit consists of the following parts (9 samples):

[0048] (1) 1.0mL 2×Reaction Mix Buffer, containing the following components:

[0049]

[0050] (2) Detection primers: upstream primer C-F: 5'-CGCAGCGGGATCATCCCAATC-3', downstream primer C-R: 5'-CGATGTGTATTTCATATAGTG-3', the concentration is 10 μM, the upstream and downstream primers are mixed, 400 μL.

[0051] (3) Taq enzyme 5U / μL.

[0052] (4) Sterilized ddH 2 O 1.0 mL.

[0053] (5) Positive control solution: crucian carp herpesvirus type 2 g...

Embodiment 2

[0071] Embodiment two: crucian carp herpesvirus type 2 PCR detection method

[0072] Using the kit described in Example 1, proceed as follows:

[0073] (1) Take 50 mg of the sample to be tested, add 600 μL of sterilized double-distilled water, grind it thoroughly with a glass homogenizer, place it in a -20 ° C refrigerator for 3 times, and centrifuge at 6000 rpm for 10 minutes at low temperature, take the supernatant, Add 200μL Tris-saturated phenol, shake and mix well, let stand for 5 minutes, then centrifuge at 12000rpm for 5 minutes, take the supernatant and add the same amount of phenol: chloroform: isoamyl alcohol for extraction twice, take the supernatant and add 2 times volume of isopropanol, after mixing, let stand for 10 minutes, centrifuge at 12000rpm for 10 minutes, remove the supernatant, add 1mL of pre-cooled 75% ethanol, let stand for 5 minutes, centrifuge at 12000rpm for 10 minutes, remove the supernatant, and place After drying in a vacuum oven, use 100 μL of ...

Embodiment 3

[0078] Example 3: Specificity experiment of crucian carp herpesvirus type 2 PCR rapid detection kit

[0079] Using the kit described in Example 1, proceed as follows:

[0080] (1) The extracted carp herpesvirus type 2, carp herpesvirus type 1, carp herpesvirus type 3, grass carp hemorrhagic disease virus, mandarin fish rhabdovirus, red sea bream iridescent virus, viral neuronecrosis virus, viral hemorrhage The nucleic acid of the sepsis virus was used as a template for PCR detection.

[0081] (2) Take 12.5 μL of 2× reaction mixture buffer, 0.5 μL of upstream and downstream primers (C-F, C-R), 0.5 μL of Taq DNA polymerase, ddH 2 O 8 μL, template 3.0 μL. After mixing evenly, centrifuge for a few seconds and place on a PCR reaction instrument.

[0082] (3) Perform PCR amplification under the following conditions: 95°C for 5 min, 1 cycle; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min, and finally store at 4°C.

[0083] (4) After the reaction is over, ...

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Abstract

The invention belongs to the technical field of reproduction, and particularly relates to a CyHV-2 (cyprinid herpesvirus 2) specificity PCR (polymerase chain reaction) detection kit and a detection method. The CyHV-2 specificity PCR detection kit comprises 2*reaction mixing buffer solutions (1.0mL), preferably designed upstream and downstream primers, Taq DNA (deoxyribonucleic acid) polymerase, positive contrast liquid, negative contrast liquid and ddH2O. The invention overcomes defects of the existing CyHV-2 detection method, and provides the novel PCR fast detection kit and the detection method. The CyHV-2 specificity PCR detection kit and the detection method have the advantages that the clinical diagnosis of CyHV-2 is feasible, in addition, the advantages of high speed, accuracy and specificity are realized, the clinical diagnosis requirements are met, and convenient conditions are provided for CyHV-2 detection.

Description

technical field [0001] The invention relates to the field of breeding technology, in particular to a genetic detection reagent and detection method for crucian carp herpes virus type 2, which is suitable for epidemiological monitoring of crucian carp carp herpes virus type 2, detection of carp herpes virus type 2 and genetic engineering technology and other fields. Background technique [0002] Cyprinid herpesvirus 2 (CyHV-2), also known as Herpesviral haematopoietic necrosis virus (HVHNV) or Goldfish haematopoietic necrosis virus (GFHNV). The virus was isolated and identified from goldfish cultured in Japan for the first time in 1992-1993, and it was also the first report of the disease. It once caused huge economic losses to the goldfish farming industry in Japan, with a mortality rate as high as 100%. The virus first appeared in the Jiangsu area of ​​my country in 2009. The blood of dying fish would flow out along the gill filaments. Fishermen called it "gill bleeding". ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2545/113
Inventor 雷燕肖洋卢刚张文文张会军马家好
Owner 广州利洋水产科技股份有限公司
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