Tilapia Lake Virus (TiLV) specificity RT-PCR detection kit and detection method

A detection kit and specific technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as no research reports on Luohu virus detection methods, and achieve rapid detection and detection. Accurate and high detection sensitivity

Inactive Publication Date: 2018-03-06
广州利洋水产科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] There is no research report o

Method used

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  • Tilapia Lake Virus (TiLV) specificity RT-PCR detection kit and detection method
  • Tilapia Lake Virus (TiLV) specificity RT-PCR detection kit and detection method
  • Tilapia Lake Virus (TiLV) specificity RT-PCR detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Luohu virus-specific RT-PCR detection kit

[0049] All chemical reagents and primers of the Luohu virus-specific RT-PCR 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.

[0050] The kit consists of the following parts (12 samples):

[0051] (1) 100 μL of 5× reverse transcription buffer;

[0052] (2) 25 μL of reverse transcriptase;

[0053] (3) 25 μL of random primers;

[0054] (4) 1.0 mL of 2× polymerase chain reaction mixed buffer, containing the following components:

[0055]

[0056] (5) Preferred specific detection primers: according to the nucleic acid sequence of Luohu virus, design specific detection primers for detecting Luohu virus, and screen the designed primer pairs with very high sensitiv...

Embodiment 2

[0075] Embodiment 2: Luohu virus-specific RT-PCR detection method uses the kit described in Embodiment 1, and proceeds according to the following steps:

[0076] (1) Take 50 mg of gills, liver, spleen, and kidney tissue samples of tilapia to be tested, add 600 μL of sterilized double-distilled water, grind them thoroughly with a glass homogenizer, and place them in a -20°C refrigerator for 3 times. Centrifuge at 6000rpm for 5 minutes at low temperature, 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.

[0077] (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,...

Embodiment 3

[0081] Example 3: Specificity experiment of Luohu virus-specific RT-PCR quick test kit

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

[0083] (1) Extracted Luohu virus, healthy tilapia, wild tilapia, koi herpes virus, carp herpes virus type 2, red sea bream iridescent virus, infectious spleen and kidney necrosis virus, grass carp hemorrhagic disease virus, carp spring The nucleic acids of viremia virus and nerve necrosis virus were detected by (RT-)PCR.

[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 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;

[0085] (3) Using a 25 μL PCR reaction system, add 12.5 μL of 2...

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Abstract

The invention belongs to the technical field of aquaculture and in particular relates to a tilapia lake virus (TiLV) specificity RT-PCR detection kit and a detection method. The TiLV reverse transcription-polymerase chain reaction rapid detection kit comprises 5*reverse transcription buffer solution, reverse transcriptase, a random primer, 2*reaction mixed buffer solution, preferred designed upstream and downstream primers, Taq DNA polymerase, positive control solution, negative control solution and ddH2O. The invention overcomes the defects of an existing TiLV detection method and provides anovel reverse transcription-polymerase chain reaction rapid detection kit and a detection method; and the detection kit and the detection method are not only practicable in TiLV clinical diagnosis butalso have the advantages of speediness, accuracy, specificity and sensitivity, clinical diagnosis requirements are met, and convenient conditions are provided for TiLV detection.

Description

technical field [0001] The invention belongs to the technical field of aquaculture, and relates to a specific detection method for tilapia luohu virus, in particular to a method for detecting tilapia luohu virus using reverse transcription-polymerase chain reaction (abbreviated RT-PCR) technology . Background technique [0002] Tilapia Lake Virus (TiLV) is an emerging virus discovered and reported in recent years. The virus was first discovered in Israel in 2009 and has been confirmed in Colombia, Ecuador, Egypt, Israel and Thailand. Although the pathogen does not cause a public health problem, it can cause significant mortality in infected populations. According to reports, the virus is highly contagious and has caused mass death of tilapia cultured in Israel, Ecuador, Egypt and other countries, with a mortality rate as high as 70%. [0003] According to the news released by the Food and Agriculture Organization of the United Nations, Luohu virus may have a wider distribu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2521/107
Inventor 雷燕肖洋张文文王娟卢刚马家好
Owner 广州利洋水产科技股份有限公司
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