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Visual rapid detection kit for KHV (Koi herpesvirus)

A koi herpes virus and detection kit technology, which is applied in the field of pathogen detection in marine aquaculture, can solve the problems of cumbersome and complicated, hindering the popularization and application of LAMP detection technology, and false positives

Inactive Publication Date: 2018-12-28
天津市水生动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this technology has been applied to the detection of KHV, but due to the cumbersome and complicated operation of the nucleic acid extraction process and the possible false positives caused by the use of dyes such as SYB-Green, it hinders the promotion and application of LAMP detection technology in the breeding process

Method used

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  • Visual rapid detection kit for KHV (Koi herpesvirus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] A koi herpes virus visualization rapid detection kit, comprising:

[0034] Reagent A: tissue lysate Ⅰ;

[0035] Reagent B: tissue lysate II;

[0036] Reagent C: isopropanol;

[0037] Reagent D: 70% ethanol water solution by volume;

[0038] Reagent E: sterile double distilled water;

[0039] Reagent F: 100mg nucleic acid extraction magnetic beads resuspended in 1ml double distilled water;

[0040] Reagent G: koi herpes virus positive plasmid;

[0041] Detection tube with reagent H: 2.5 μl of 10×Buffer, 3.5 μl of 40 mM deoxyribonucleotide, 100 mM MgSO in the detection tube 4 3 μl, 5M betaine 1 μl, 100 μM primer KHV-FIP shown in SEQ ID NO.1 0.4 μl, 100 μM primer KHV-BIP shown in SEQ ID NO.2 0.4 μl, 100 μM primer KHV-BIP shown in SEQ ID NO.2 0.2 μl of primer KHV-LF shown in .3, 0.2 μl of primer KHV-LB shown in SEQ ID NO.4 of 100 μ M, use of 0.05 μ l of primer KHV-F3 shown in SEQ ID NO.5 of 100 μ M 0.05 μl of primer KHV-B3 shown in SEQ ID NO.6, 1 μl of 8 U / μl Bst DN...

Embodiment 2

[0044] A koi herpes virus visualization rapid detection kit, comprising:

[0045] It is the 70% ethanol water solution that the volume percentage concentration of reagent D of embodiment 1 is replaced by the volume percentage concentration of 65% ethanol water solution;

[0046] The 100 mg nucleic acid extraction magnetic beads in the reagent F of Example 1 were replaced with 90 mg nucleic acid extraction magnetic beads; the others were the same as in Example 1.

Embodiment 3

[0048]A koi herpes virus visualization rapid detection kit, comprising:

[0049] It is the 70% ethanol aqueous solution that the volume percent concentration of the reagent D of embodiment 1 is replaced by the 80% ethanol aqueous solution with the volume percent concentration;

[0050] The 100 mg nucleic acid extraction magnetic beads in the reagent F of Example 1 were replaced with 110 mg nucleic acid extraction magnetic beads; the others were the same as in Example 1.

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Abstract

The invention discloses a visual rapid detection kit for KHV (Koi herpesvirus). The visual rapid detection kit comprises a reagent A, a reagent B, a reagent C, a reagent D, a reagent E, a reagent F, areagent G and a detection tube containing a reagent H, wherein the reagent A is a tissue lysate I; the reagent B is a tissue lysate II; the reagent C is isopropyl alcohol; the reagent D is aqueous ethanol with volume percentage concentration being 65%-80%; the reagent E is sterilized double distilled water; the reagent F is 90-110 mg of nucleic acid extracting magnetic beads resuspended in 1 ml of double distilled water; the reagent G is KHV positive plasmid. The operation process of the kit is simple, KHV detection can be completed in 1.5-2 h, and the KHV detection can be completed only by one water bath kettle or metal bath without any high-end instruments and equipment.

Description

technical field [0001] The invention relates to a detection technology for pathogens in marine aquaculture, in particular to a detection kit for Koiherpersvirus (KHV). Background technique [0002] Koi herpersvirus (KHV), also known as CyHV-Ⅲ, is the main pathogen of koi herpes virus disease, with strong infectivity and high mortality. The virus was first discovered in Israel in 1998, and then broke out in large areas in the United States, Indonesia, Europe, Japan and other countries. It also occurred in Guangdong, Liaoning, Jilin, North China and other places in China. KHV is a double-stranded DNA virus with a diameter of about 170-230nm. It contains 31 viral polypeptides and has a core, capsid and envelope structure. The nucleocapsid is a symmetrical icosahedron with a diameter of 100-110nm. KHV mainly infects koi, common carp and their common variants. Fish latently infected with KHV do not show clinical symptoms, and may break out and cause death when the water temperat...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12N15/10
CPCC12N15/1013C12Q1/686C12Q1/705
Inventor 薛淑霞孙妍董学旺杨海涛李晶晶王雪惠魏俊利
Owner 天津市水生动物疫病预防控制中心
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