Eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method

A technology of Chinese mitten crab and detection kit, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, and can solve the problems of unseen rapid and accurate methods for detecting Chinese mitten crab. , to achieve the effect of fast detection, high efficiency and high detection sensitivity

Inactive Publication Date: 2015-10-21
GUANGZHOU JINSHUI ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no fast and accurate method for detecting Chinese mitten crabs. In order to find out whether the cultured Chinese mitten crabs are infected by white spot virus as early as possible, it is necessary to establish a fast, accurate and sensitive detection method.

Method used

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  • Eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method
  • Eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method
  • Eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Chinese mitten crab white spot virus PCR rapid detection kit

[0045] All chemical reagents and primers of the Chinese mitten crab white spot virus PCR rapid 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.

[0046] The kit consists of the following parts (9 samples):

[0047] (1) 1.0 mL of 2× reaction mixture buffer, containing the following components:

[0048]

[0049] (2) Detection primers: upstream primer WSSV-F: 5'-GTGTACTAGGAATATTGGAAT-3', downstream primer WSSV-R: 5'-CGGCATTCTTCATGGCTTCTG-3', the concentration is 10 μM, the upstream and downstream primers are mixed, 400 μL.

[0050] (3) Taq enzyme 5U / μL.

[0051] (4) Sterilized ddH 2 O 1.0 mL.

[0052] (5) Positive control solution: Genomic D...

Embodiment 2

[0070] Embodiment two: Chinese mitten crab white spot virus PCR detection method

[0071] Using the kit described in Example 1, proceed as follows:

[0072] (1) Take 50 mg of the sample to be tested, add 600 μL of sterilized double-distilled water, grind it thoroughly with a glass homogenizer, place it in a -20 ° C refrigerator for 3 times, and centrifuge at 6000 rpm for 10 minutes at low temperature, take the supernatant, Add 200μL Tris-saturated phenol, shake and mix well, let stand for 5 minutes, then centrifuge at 12000rpm for 5 minutes, take the supernatant and add the same amount of phenol: chloroform: isoamyl alcohol for extraction twice, take the supernatant and add 2 times volume of isopropanol, after mixing, let stand for 10 minutes, centrifuge at 12000rpm for 10 minutes, remove the supernatant, add 1mL of pre-cooled 75% ethanol, let stand for 5 minutes, centrifuge at 12000rpm for 10 minutes, remove the supernatant, and place After drying in a vacuum oven, use 100 μ...

Embodiment 3

[0077] Example 3: specificity experiment of PCR rapid detection kit for mitten crab white spot disease virus

[0078] Using the kit described in Example 1, proceed as follows:

[0079] (1) Using the extracted nucleic acid of white spot virus, spiroplasma, Aeromonas hydrophila, infectious spleen and kidney necrosis virus, mandarin fish rhabdovirus respectively as templates, carry out PCR detection.

[0080] (2) Take 10 μL of 2× reaction mixture buffer, 0.5 μL of upstream and downstream primers (WSSV-F, WSSV-R), 0.5 μL of Taq DNA polymerase, ddH 2 O 5.5 μL, template 3.0 μL. After mixing evenly, centrifuge for a few seconds and place on a PCR reaction instrument.

[0081] (3) Perform PCR amplification under the following conditions: 95°C for 5 min, 1 cycle; 95°C for 30 s, 56°C for 30 s, 72°C for 40 s, 30 cycles; 72°C for 10 min, and finally store at 4°C.

[0082] (4) After the reaction is over, take 10 μL and add it to 2 μL bromophenol blue and mix it evenly. After electrophor...

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Abstract

The invention relates to the technical field of breeding, in particular to an eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method. The eriocheir sinensis white spot syndrome virus specificity PCR detection kit comprises 1.0 mL of a 2*reaction mixture buffer solution, preferably-designed upstream or downstream primers, Taq DNA polymerase, positive control fluid, negative control fluid and ddH2O. The novel and rapid PCR detection kit and method overcome the defects of an existing eriocheir sinensis white spot syndrome virus detection method. The eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method are practicable for clinic diagnosis on the eriocheir sinensis white spot syndrome virus, have the advantages of being rapid, accurate and special, meet the requirement of clinic diagnosis, and provide convenience for eriocheir sinensis white spot syndrome virus detection.

Description

technical field [0001] The invention relates to the field of breeding technology, in particular to a specific PCR rapid detection kit and detection method for the pathogenic leukoplakia virus that causes the pathogenic disease of mitten crab sinensis, and in particular to a gene detection reagent and a detection method for the pathogenic leukoplakia virus of the mitten crab sinensis It is suitable for epidemiological monitoring of Chinese mitten crab white spot disease virus, detection of white spot disease virus and genetic engineering technology and other fields. Background technique [0002] White spot disease virus (WSSV) is a virus that seriously endangers the current shrimp farming industry. It mainly causes white spot lesions on the inner side of the shrimp carapace and leads to a large number of deaths. It has a wide range of prevalence and has sensitive hosts in crustaceans, copepods and insects. [0003] Chinese mitten crab, also known as river crab, is an importa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2565/125
Inventor 雷燕肖洋张文文张会军王娟卢刚马家好
Owner GUANGZHOU JINSHUI ANIMAL HEALTH PROD
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