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RT-PCR (reverse transcription-polymerase chain reaction) detection kit for spring viremia of carp virus as well as detection method

A carp spring viremia, RT-PCR technology, applied in the field of aquaculture, to achieve the effect of accurate detection, high detection sensitivity and rapid detection

Inactive Publication Date: 2018-05-08
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

[0004] The traditional methods of diagnosing carp spring viremia virus are virus isolation, cell culture, electron microscope observation and immunoassay, etc., lack of rapid, accurate and sensitive detection methods for the virus

Method used

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  • RT-PCR (reverse transcription-polymerase chain reaction) detection kit for spring viremia of carp virus as well as detection method
  • RT-PCR (reverse transcription-polymerase chain reaction) detection kit for spring viremia of carp virus as well as detection method
  • RT-PCR (reverse transcription-polymerase chain reaction) detection kit for spring viremia of carp virus as well as detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Reverse transcription-polymerase chain reaction rapid detection kit for carp spring viremia virus

[0047] All chemical reagents and primers of the reverse transcription-polymerase chain reaction rapid detection kit for carp spring viremia virus 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.

[0048] The kit consists of the following parts (12 samples):

[0049] (1) 100 μL of 5× reverse transcription buffer;

[0050] (2) 25 μL of reverse transcriptase;

[0051] (3) 25 μL of random primers;

[0052] (4) 1.0 mL of 2× polymerase chain reaction mixed buffer, containing the following components:

[0053]

[0054] (5) Preferred specific detection primers: according to the nucleic acid sequence of carp spring viremia vi...

Embodiment 2

[0081] Example 2: Rapid detection method for carp spring viremia virus by reverse transcription-polymerase chain reaction

[0082] Using the kit described in Example 1, proceed as follows:

[0083] (1) Take 50 mg of the liver, spleen, kidney and other tissue samples of the fish to be tested, add 600 uL of sterilized double distilled water, grind them thoroughly with a glass homogenizer, and place them in a -20°C refrigerator for 3 times of freezing and thawing at 6000rpm Centrifuge at low temperature for 5 minutes, take the supernatant, use the traditional Trizol method or a commercial RNA extraction kit to extract RNA, and use ddH 2 O is dissolved, which is the RNA template for the test.

[0084] (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, Put it on ...

Embodiment 3

[0088] Example 3: Reverse transcription-polymerase chain reaction rapid detection kit specificity experiment for carp spring viremia virus

[0089] Using the kit described in Example 1, proceed as follows:

[0090] (1) Carp spring viremia virus, koi herpes virus, grass carp hemorrhagic disease virus, channel catfish herpes virus, crucian carp herpes virus type 2, mandarin fish rhabdovirus, viral hemorrhagic septicemia virus, red sea bream iridescent virus were extracted respectively The nucleic acid was detected by RT-PCR.

[0091] (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, Put it on a PCR machine, reverse transcription reaction at 37°C for 15min, inactivate reverse transcriptase at 85°C for 5sec, store at 4°C, and obtain cDNA product;

[0092] (3) U...

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Abstract

The invention relates to the technical field of aquaculture, and in particular discloses a reverse transcription-polymerase chain reaction (RT-PCR) detection kit for spring viremia of carp virus. TheRT-PCR detection kit for the spring viremia of carp virus comprises a 5*reverse transcription buffer solution, reverse transcriptase, a random primer, a 2*reaction mixed buffer solution, preferably designed upstream and downstream primers, Taq DNA polymerase, positive control liquid, negative control liquid and ddH2O. The invention provides a novel RT-PCR rapid detection kit and a detection methodso as to overcome the defects in the existing method for detecting the spring viremia of carp virus. Clinical diagnosis on the spring viremia of carp virus is practicable, the advantages of rapidness, accuracy and specificity are achieved, the requirements of clinical diagnosis are met and convenient conditions are providing for detecting the spring viremia of carp virus.

Description

technical field [0001] The invention belongs to the technical field of aquaculture, and relates to a method for detecting carp spring viremia virus, in particular to a method for detecting carp spring viremia virus by using reverse transcription-polymerase chain reaction (RT-PCR for short) technology . Background technique [0002] Carp spring viremia is a disease caused by Spring Viremia of Carp Virus (SVCV) of Rhabdoviridae genus Vesivirus, which is mainly spread in carp culture in Europe and caused Huge economic loss is one of the diseases that need to be declared to OIE as stipulated by the World Organization for Animal Health (OIE). In recent years, our investigation on carp diseases found that SVCV is also prevalent in carp cultured in my country. [0003] The SVCV virion is bullet-shaped, which is a typical morphological feature of rhabdoviruses, with a size of (90-180) nm×(60-90) nm and a genome length of 11019 nucleotides. Carp is the most susceptible host of SVC...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2521/107
Inventor 雷燕肖洋王娟张文文卢刚马家好
Owner 广州利洋水产科技股份有限公司
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