RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and RT-LAMP detection method for SVCV (spring viremia of carp virus)

A technology of RT-LAMP and spring carp viremia, which is applied in the field of RT-LAMP detection kits for spring carp viremia virus, can solve the problems of relatively high requirements on instruments and personnel, complicated procedures, long time consumption, etc., so as to improve the sensitivity , The detection cost is low, the specificity is good

Inactive Publication Date: 2013-01-16
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

The "gold standard" for SVCV detection is to isolate the virus through cell culture and identify it using immunological techniques or molecular biology techniques. This process is time-consuming and complicated.
In addition, it is difficult to prepare high-titer antiserum, which also limits the use of immunological methods
OIE Aquatic Animal Disease Diagnosis Manual, my country's national standard and industry standard for SVCV detection have listed RT-PCR as a diagnostic method, but RT-PCR method is more complicated to operate, requires relatively high equipment and personnel, and is not suitable for on-site rapid detection. Detection and grass-roots popularization and application

Method used

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  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and RT-LAMP detection method for SVCV (spring viremia of carp virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A carp spring viremia virus RT-LAMP detection kit comprises the following components:

[0043] The kit includes the following components: 10×ThermoPol Reaction Buffer; Bst DNA polymerase (NEB); dNTPs; AMV reverse transcriptase (BBI); outer primers F3 and B3, inner primers FIP and BIP, Betaine (Sigma), MgCl 2 , DTT (Invitrogen), 1000 x SYBR Green I (Invitrogen).

[0044] F3: 5, -GGGATAGCTTCGGACACAAG-3,

[0045] B3:5,-CCATCAGCAAAGTCCGGTAT-3,

[0046] FIP: 5, -GTTTCCCATCTGGTAGCCCGTCTTTTTAGATCGGGCCTTTTAACCTG-3,

[0047] BIP: 5'-AGGTGAGTGCTGAGGATGATGCTTTTTTGCCCTTCCCACTCTGT-3'.

Embodiment 2

[0049] Optimization of different concentrations of Mg2+ in carp spring viremia virus RT-LAMP detection kit

[0050] 1. Take the sample to be tested and extract the virus RNA:

[0051] Culture carp epithelial papilloma cells (EPC cells) to a confluent monolayer, suck out the culture medium, and inoculate 1 mL of carp spring viremia virus (Zhang Peng, Liu Hong, Chen Xiaoxuan, et al. Preparation and Characterization of Monoclonal Antibody to Carp Spring Viremia Virus[J], Chinese Journal of Preventive Veterinary Medicine, 2011, 33(4): 305-308.) Cytotoxic material, adding 10 μL of Polybrene (final concentration 10 μg / ml), placed in a 26°C incubator for 1 hour to allow the virus to adsorb to the cells, and gently shake the culture bottle once every 20 minutes during the adsorption process to make the virus liquid fully and evenly contact with the cell monolayer. After 1 hour of adsorption, Aspirate and discard the virus liquid, add 5mL of 2% fetal bovine serum (V / V) culture solutio...

Embodiment 3

[0060] Optimization of the reaction temperature of RT-LAMP detection method for carp spring viremia virus:

[0061] 1. Take the sample to be tested and extract the virus RNA:

[0062] Harvest the EPC cells infected with SVCV 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 250 μl of the supernatant, and use LS reagent was used to extract RNA according to the instructions, and finally dissolved in 50 μl sterilized water, and stored at -80°C for later use.

[0063] 2. The reaction system of RT-LAMP amplification:

[0064] A 25 μl reaction system was used, including: 0.8 μM each of the inner primers FIP and BIP, 0.1 μM of the outer primers F3 and B3, dNTPs 1 mM, Betaine 0.5 M, DTT 4 mM, MgCl 2 8mM, Bst DNA polymerase 8U, AMV reverse transcriptase 5U, template RNA 5μl, 10×ThermoPol Reaction Buffer 2.5μl.

[0065] 3. Reaction conditions for RT-...

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Abstract

The invention discloses a RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and a RT-LAMP detection method for SVCV (spring viremia of carp virus). The RT-LAMP detection kit of SVCV comprises 10*ThermoPol Reaction buffer, Bst DNA (deoxyribonucleic acid) polymerase, dNTPs (deoxynucleoside triphosphates), AMV reverse transcriptase, outer primer F3 and B3, inner primer FIP, BIP and Betaine, MgC 12, DTT, and 1000*SYBR Green I. The RT-LAMP detection kit is simple, fast, and high in specificity and sensitivity, only needs a water bath kettle or a metal bath to accurately detect SVCV in samples in 2 hours, can detect SVCV of infected tissues of sick fish, can detect cells infected by SVCV, and is suitable for fast field detection of SVCV.

Description

technical field [0001] The invention belongs to the technical field of fish virus detection, in particular to a carp spring viremia virus RT-LAMP detection kit, and also relates to a carp spring viremia virus RT-LAMP detection kit for detecting carp spring viremia way of the virus. Background technique [0002] Spring viraemia of carp virus (SVCV), a tentative species belonging to the order Mononegavirales, the family Rhabdoviridae, and the genus Vesiculovirus, Currently there is only one serotype. Spring Viraemia of Carp (SVC) caused by SVCV is an acute, hemorrhagic and highly contagious epidemic that seriously endangers fishery production and can cause devastating blows. The World Organization for Animal Health (OIE) lists it as an important disease that must be reported. In 2008, my country's newly promulgated list of animal diseases listed it as a first-class animal disease. It is the first and only fish virus disease that has been listed as a first-class infectious d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 张辉曾令兵周勇范玉顶徐进
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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