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Detection kit and detection method for siniperca chuatsi rhabdoviruses

A detection kit and rhabdovirus technology, applied in 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: 2014-09-10
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 for siniperca chuatsi rhabdoviruses
  • Detection kit and detection method for siniperca chuatsi rhabdoviruses
  • Detection kit and detection method for siniperca chuatsi rhabdoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment one: the reverse transcription-polymerase chain reaction detection kit of mandarin fish rhabdovirus

[0033] All chemical reagents and primers of the reverse transcription-polymerase chain reaction detection kit for mandarin fish rhabdovirus 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.

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

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

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

[0037] (3) 25 μL of random primers;

[0038] (4) 1.0 mL of 2× polymerase chain reaction mixed buffer, containing the following components:

[0039]

[0040](5) Design and synthesis of detection primers: According to the gene sequence of mandarin fish rhabdovirus rele...

Embodiment 2

[0059] Embodiment two: the reverse transcription-polymerase chain reaction detection method of mandarin fish rhabdovirus

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

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

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

Embodiment 3

[0066] Embodiment three: the specificity experiment of reverse transcription-polymerase chain reaction rapid test kit of mandarin fish rhabdovirus

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

[0068] (1) RT-PCR detection was carried out with the extracted RNA of infectious hematopoietic organ necrosis virus, viral hemorrhagic sepsis virus, flounder rhabdovirus, carp spring viremia virus and barracuda rhabdovirus respectively.

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

[0070] (3) Using a 25 μL PCR reaction system, add 12.5 μL of 2× reaction mixture buf...

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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 for mandarin fish rhabdovirus, in particular to a method for detecting mandarin fish rhabdovirus by reverse transcription-polymerase chain reaction (RT-PCR for short). way of the virus. Background technique [0002] In recent years, large-scale outbreaks of epidemic diseases have occurred in Foshan and Zhongshan in Guangdong Province, Jining in Shandong, and snakehead, mandarin fish, and California perch farms in Hubei and other places, causing huge economic losses. The laboratory detected and analyzed the diseased fish from both bacteriological and virological aspects, and confirmed that the disease causing the explosive death of these farmed fish was rhabdovirus disease, and the pathogen was Siniperca chuatsi rhabdovirus (SCRV) . [0003] Rhabdovirus (Rhabdovirus) is a single-strand non-segmented negative-strand RNA...

Claims

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Application Information

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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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