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LAMP detection kit for Koi herpesvirus and detection method

A technology of koi herpes virus and detection kit, which is applied in the field of fish virus detection to achieve the effect of improving sensitivity, simple identification and good specificity

Inactive Publication Date: 2013-04-24
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have their own limitations. In order to detect and diagnose koi herpes virus disease early, it is necessary to establish an accurate, fast and sensitive detection me...

Method used

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  • LAMP detection kit for Koi herpesvirus and detection method
  • LAMP detection kit for Koi herpesvirus and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A koi herpes virus LAMP detection kit, comprising the following components:

[0044] The kit includes the following components: 10×ThermoPol Reaction Buffer; Bst DNA polymerase (NEB); dNTPs; outer primers F3 and B3, inner primers FIP and BIP, Betaine (Sigma), MgCl 2 , 1000 x SYBRGreenI (Invitrogen).

[0045] F3: 5'-GCATCGCCGTCAAGCAC-3';

[0046] B3: 5'-GCAGCTGCACGACTCC-3';

[0047] FIP: 5'-AGATGGCCGGGTAGGTCGCTTTTGCCATAGACCAGCGCTACA-3';

[0048] BIP: 5'-ACCTGTACGAGGTGATGCAGCTTTAGGTCGGGGAAGAACTGTC-3'.

Embodiment 2

[0050] Koi Herpes Virus LAMP Detection Kit Different Concentrations of Mg 2+ Optimization

[0051] 1. Take the sample to be tested and extract the virus DNA:

[0052] Cultivate the koi fin ray cell line (Koi-Fin) to a confluent monolayer, suck out the culture medium, and inoculate 1 mL of koi herpes virus cytotoxic material (Zhu Xia, Li Xinwei, Wang Hao, et al. Isolation and identification of a koi herpes virus strain[J]. Chinese Journal of Preventive Veterinary Medicine, 2011,33(5):340-343. Add 10μL Polybrene (final concentration 10μg / ml ), placed in a 26°C incubator for 1 hour to allow the virus to adsorb the cells, and gently shake the flask every 20 minutes during the adsorption process to make the virus liquid fully and evenly contact the cell monolayer. After 1 hour of adsorption, discard the virus liquid and add 5mL of 2% fetal bovine serum (V / V) culture medium, cultured at 26°C until the cells showed obvious cytopathic effect, collected cytopathic materials, and ...

Embodiment 3

[0061] Optimization of reaction temperature for koi herpesvirus LAMP detection method:

[0062] 1. Take the sample to be tested and extract the virus DNA:

[0063] Harvest the KHV-infected Koi-Fin cells after 90% of the lesions appear (the preparation method is the same as in Example 2), freeze and thaw three times at -80°C to room temperature, centrifuge at 5000r / min for 30min, take 250μl of the supernatant, and use Viral DNA Kit The kit was used to extract DNA according to the instructions, and finally dissolved in 50 μl of sterilized water, and stored at -20°C for later use.

[0064] 2. The reaction system of LAMP amplification:

[0065] A 25 μl reaction system was used, including: 0.8 μM each of the inner primers FIP and BIP, 0.1 μM each of the outer primers F3 and B3, dNTPs1 mM, Betaine0.5M, MgCl 2 8mM, Bst DNA polymerase 8U, template DNA 5μl, 10×ThermoPolReaction Buffer 2.5μl, deionized water to make up the balance.

[0066] 3. Reaction conditions for LAMP amplifica...

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Abstract

The invention discloses an LAMP detection kit for Koi herpesvirus and a detection method. The LAMP detection kit comprises a 10*ThermoPol Reaction buffer liquor, Bst DNA (Deoxyribose Nucleic Acid) polymerase, dNTPs, outer primers F3 and B3, inner primers FIP and BIP, Betaine, MgCL2, and 1000*SYBR Green I. The LAMP detection kit provided by the invention has the characteristics of simplicity, convenience, rapidness and high specificity and sensitivity, and KHV (Koi Herpesvirus) in a sample can be accurately detected within 2 hours by a water bath kettle or a metal bath. The diseased fish tissue infected by KHV can be detected, and cells infected by KHV can be detected, so that the kit is very suitable for field fast detection of KHV.

Description

technical field [0001] The invention belongs to the technical field of fish virus detection, specifically relates to a koi herpes virus LAMP detection kit, and also relates to a method for detecting koi herpes virus using the koi herpes virus LAMP detection kit. Background technique [0002] In May 1998, Koi herpes virus disease first broke out in the Magan Michael area of ​​Israel. In the same year, the pathogenic Koi herpes virus (Koi herpes virus, KHV) was successfully isolated. herpesvirus), also known as carp herpesvirus type III (CyHV-III). Koi herpes virus disease spread rapidly throughout the world. In 2002, it was confirmed that the disease had spread to my country for the first time. In recent years, it has caused serious economic losses to the aquaculture industry of Chinese carp and carp. [0003] The current diagnostic methods include cell culture separation technology, electron microscope technology, PCR, ELISA, in situ hybridization technology, etc. Polymera...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 曾令兵张辉周勇徐进范玉顶
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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