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Detection kit and detection method of mandarin fish rhabdovirus

A detection kit and rhabdovirus technology, applied to the detection of rhabdovirus in mandarin fish, using reverse transcription-polymerase chain reaction technology to detect rhabdovirus in mandarin fish, to achieve rapid detection, accurate detection and high efficiency Effect

Active Publication Date: 2017-02-15
GUANGZHOU JINSHUI ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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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  • Detection kit and detection method of mandarin fish rhabdovirus
  • Detection kit and detection method of mandarin fish rhabdovirus
  • Detection kit and detection method of mandarin fish rhabdovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Reverse transcription-polymerase chain reaction detection kit for mandarin fish rhabdovirus

[0033] All the chemical reagents and primers of the reverse transcription-polymerase chain reaction detection kit for mandarin fish rhabdovirus described in this example are purchased from professional reagent companies. However, the sources of the above-mentioned reagents and primers do not constitute any limitation to the present invention. The present invention can prepare relevant reagents and synthesize relevant primers by itself.

[0034] The kit consists of the following parts (12 samples):

[0035] (1) 5×100μL of reverse transcription buffer;

[0036] (2) 25μL of reverse transcriptase;

[0037] (3) Random primer 25μL;

[0038] (4) 2× polymerase chain reaction mixing buffer 1.0mL, containing the following ingredients:

[0039]

[0040] (5) Design and synthesis of detection primers: According to the gene sequence of mandarin fish rhabdovirus published in GenBank, design a...

Embodiment 2

[0059] Example 2: Reverse transcription-polymerase chain reaction detection method for mandarin fish rhabdovirus

[0060] Using the kit described in Example 1, proceed as follows:

[0061] (1) Take 50 mg of the sample to be tested, add 600 uL of sterilized double distilled water, fully grind it with a glass homogenizer, place it in a refrigerator at -20°C, freeze-thaw for 3 times, centrifuge at 6000 rpm for 5 minutes, and take the supernatant. Use traditional Trizol method or commercial RNA extraction kit to extract RNA, use ddH 2 O dissolved, which is the RNA template for the test.

[0062] (2) Take 7μL of the RNA template of the sample to be tested and add it to the PCR reaction tube. Then add 2μL of 5× reverse transcription buffer, 0.5μL of reverse transcriptase, 0.5μL of random primers, and the total reaction volume is 10μL. After mixing well, Place it on a PCR machine, perform reverse transcription reaction at 37°C for 15 minutes, and inactivate reverse transcription at 85°C fo...

Embodiment 3

[0066] Example 3: Specific experiment of reverse transcription-polymerase chain reaction rapid test kit for mandarin fish rhabdovirus

[0067] Using the kit described in Example 1, proceed as follows:

[0068] (1) The extracted RNAs of infectious hematopoietic organ necrosis virus, viral hemorrhagic sepsis virus, flounder rhabdovirus, carp viremia virus, and barracuda rhabdovirus were detected by RT-PCR.

[0069] (2) Take 7μL of the RNA template of the sample to be tested and add it to the PCR reaction tube. Then add 2μL of 5× reverse transcription buffer, 0.5μL of reverse transcriptase, 0.5μL of random primers, and the total reaction volume is 10μL. After mixing well, Place it on a PCR machine, perform reverse transcription reaction at 37°C for 15 minutes, and inactivate reverse transcription at 85°C for 5 seconds to obtain a cDNA product;

[0070] (3) Using a 25μL PCR reaction system, add 12.5μL of 2× Reaction Mixing Buffer to 0.2mL PCR reaction tube, each 0.5μL of upstream primer P...

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Abstract

The invention belongs to the technical field of detection and diagnosis of aquatic animal pathogeny, and discloses a detection kit and detection method for siniperca chuatsi rhabdoviruses. The reverse transcription-polymerase chain reaction type rapid detection kit for the siniperca chuatsi rhabdoviruses is filled with a 5* reverse transcription buffer solution, reverse transcriptase, a random primer, 1.0 mL of 2*reaction mixed buffer solution, a preferably-designed sense primer, a preferably-designed reverse primer, Taq DNA polymerase, a positive control solution, a negative control solution and ddH2O. The novel reverse transcription-polymerase chain reaction type rapid detection kit and detection method for the siniperca chuatsi rhabdoviruses overcome shortcomings of an existing detection method for the siniperca chuatsi rhabdoviruses, are practicable when applied to clinical diagnosis of the siniperca chuatsi rhabdoviruses, have the advantages of rapidity, accuracy and specificity, meet requirements of the clinical diagnosis, and provide convenience for detection of the siniperca chuatsi rhabdoviruses.

Description

Technical field [0001] The invention belongs to the technical field of detection and diagnosis of aquatic animal pathogens, and relates to a detection method of mandarin fish rhabdovirus, in particular to a detection method of mandarin fish rhabdovirus using reverse transcription-polymerase chain reaction (RT-PCR) technology for short Virus method. Background technique [0002] In recent years, large-scale epidemic diseases have broken out in the farms of snakehead, mandarin fish, and California bass in Foshan and Zhongshan in Guangdong Province, Jining in Shandong, and Hubei and other places, causing huge economic losses. The laboratory tested and analyzed the diseased fish from both bacteriology and virology, and confirmed that the disease that caused the outbreak of these farmed fishes was rhabdovirus disease, and its pathogen was Siniperca chuatsi rhabdovirus (SCRV) . [0003] Rhabdovirus (Rhabdovirus) is a single-stranded unsegmented negative-strand RNA virus with a size of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2531/113
Inventor 雷燕张会军刘婉王惠儒张文文
Owner GUANGZHOU JINSHUI ANIMAL HEALTH PROD
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