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Frog virogene diagnostic kit and detecting method thereof

A gene diagnosis and kit technology, which is applied in the field of kits and detection for frog virus gene diagnosis, can solve the problems of troublesome material preparation, difficult to popularize, restrict application and development, etc., so as to avoid the spread of the virus and improve the efficiency of scientific management. , the effect of high practical value

Inactive Publication Date: 2005-01-26
SUN YAT SEN UNIV
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  • Summary
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  • Description
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Problems solved by technology

Some of these detection methods have high technical requirements, long detection time, troublesome preparation of materials required, high cost, and low sensitivity and low specificity of some methods. Therefore, it is very difficult for the majority of farmers to master key technologies and it is difficult to promote , limiting the application and development of these technologies

Method used

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  • Frog virogene diagnostic kit and detecting method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the genetic diagnosis kit of frog virus

[0040] The kit consists of the following components (10 samples):

[0041] 1). Sample diluent (solution A), 2 tubes, 5ml / tube, filled with phosphate buffer (1×PBS), pH7.4.

[0042] 2). Template extract solution (solution B), self-prepared or prepared, phenol / chloroform / isoamyl alcohol, the ratio is 25:24:1.

[0043] 3). Solution C, 1 tube, 200μl / tube, filled with 5M NaCL.

[0044] 4). Liquid D, self-prepared, 1ml / part x 10parts, 10ml, mainly absolute ethanol.

[0045] 5). E liquid, self-prepared, mainly 70% ethanol.

[0046] 6). Solution F, 1 tube, 30μl / part x 10 parts, 300μl / tube, filled with sterilized double distilled water.

[0047] 7).PCR reaction solution (solution G), 1 tube, 25μl / part x 10 copies, 250μl / tube, containing PCR first expansion reaction solution (25μl system), including ddH 2 O, 10×Buffer (including mg 2+ ), dNTP, primer F1, primer R1 and TaqE.

[0048] 8). Positive control (solution H), ...

Embodiment 2

[0061] Embodiment 2: the detection method of frog virus

[0062] Use the test kit of embodiment 1, carry out according to the following steps:

[0063] 1). Take 0.1 g of the sample to be tested, add 1 ml of sample diluent (A solution) to dilute 10 times, and homogenize in an ice bath in a homogenizer.

[0064] 2). 4000r / min centrifugal 10mm.

[0065] 3). Take 600 μl of the supernatant to extract with the template extraction solution (solution B), and mix by inverting up and down several times.

[0066] 4). Centrifuge at 12000r / min for 10min.

[0067] 5). Take 500ul of supernatant and add 20ul of solution C, then add 1ml of solution D, and precipitate at -20°C for 1 hour.

[0068] 6). Centrifuge at 12000r / min for 10min.

[0069] 7). Discard the supernatant, add solution E to wash, centrifuge at 12000r / min for 5min, and wash twice.

[0070] 8). Discard the supernatant and dry it naturally or on an ultra-clean bench.

[0071] 9). Add 20-30 μl F solution to resuspend and dis...

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Abstract

The present invention is tiger frog virus gene diagnosing kit and its usage. The kit and its usage is designed based on two pairs of primers for the conservation sequence of tiger frog virus gene. By means of PCR technology, the present invention detects qualitatively the specific DNA nucleic acid segment of tiger frog virus fast, simply, specifically and sensitively. The present invention may be used in the tracing detection of different stages of tiger frog cultivation and environment monitoring.

Description

technical field [0001] The invention relates to a diagnostic kit and a detection method for diseases of aquatic economic animals, in particular to a kit and a detection method for gene diagnosis of frog virus (TFV, Tiger Frog Virus). Background technique [0002] In recent years, diseases of reptiles, amphibians and fish caused by iridoviruses have become widespread in the Americas, Europe, Asia and Australia. We found that a large number of tadpoles died in the farmed tiger frog (Ranatig rinarugulosa), and conducted research on its pathogen, and found that it was caused by an iridescent virus—tigerfrog virus (TFV for short). In many countries and regions, it has been reported that iridescent virus has caused outbreaks of aquatic economic animals, causing a large number of deaths of these animals and causing serious losses to the aquaculture industry. At present, there is no effective treatment for frog virus disease. The most effective preventive measure is to promote the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 何建国吕玲黄志坚邓敏翁少萍
Owner SUN YAT SEN UNIV
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