Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses

A technology of RT-LAMP and reovirus, applied in the field of fish virus detection, can solve the problems of inapplicable on-site rapid detection and popular application at the grassroots level, high requirements for instruments and personnel, and complicated operation, so as to achieve low detection cost and high efficiency. Good sensitivity and specificity

Inactive Publication Date: 2012-10-03
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 RT-PCR method is more complicated to operate, and has relatively high requirements for instruments and personnel, and is not suitable for on-site rapid detection and popular application at the grassroots level

Method used

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  • Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses
  • Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A grass carp reovirus RT-LAMP detection kit comprises the following components:

[0045] The kit includes the following components: 1×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).

[0046] F3: 5'-CAGTGTGATCTCGACTTCCG-3';

[0047] B3: 5'-AGACCAACGCGTCAATCG-3';

[0048] FIP: 5'-CGGTCGTCTGACGTACACCGTTTTTTGCCGGCATATGGGGTAA-3';

[0049] BIP: 5'-AGTTGGGTCAATTGGCTACGGTTTTTAGCACCATGGTACTGTTCG-3'.

Embodiment 2

[0051] Grass carp reovirus (GCRV-104) RT-LAMP detection method with different concentrations of Mg 2+ Optimization

[0052] 1. Take the sample to be tested and extract the virus RNA:

[0053] Grass carp samples suspected of suffering from grass carp hemorrhagic disease were collected in Wencun, Jiangling County, Jingzhou City, Hubei Province. The liver, spleen, kidney and other visceral tissues of the diseased fish were collected in a sterile petri dish, cut into pieces with sterile ophthalmic scissors, and added 10 Double the volume (V / W) of PBS, together with the tissue fragments, were transferred to a glass homogenizer and ground into a tissue homogenate under the condition of an ice bath. The tissue homogenate was frozen at -80°C and thawed at room temperature (20-25°C, the same up and down), and so repeated freezing and thawing 3 times. Centrifuge at 5000×g for 30 min at 4°C, and filter the supernatant with a 0.22 μm filter to sterilize. Take 1 mL of tissue homogenate ...

Embodiment 3

[0063] Optimization of reaction temperature for grass carp reovirus (GCRV) RT-LAMP detection method:

[0064] 1. Take the sample to be tested and extract the virus RNA:

[0065] The CIK cells infected with GCRV-104 were harvested after 90% of the lesions appeared (the preparation method was the same as in Example 2), frozen and thawed three times at -80°C to room temperature, centrifuged at 5000r / min for 30min, and 250 μl of the supernatant was taken and used 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.

[0066] 2. The reaction system of RT-LAMP amplification:

[0067] A 25 μl reaction system was used, including: 0.8 μM of inner primers FIP and BIP, 0.1 μM of outer primers F3 and B3, 1 mM of dNTPs, 0.5 M of Betaine, 4 mM of DTT, MgCl 2 8mM, Bst DNA polymerase 8U, AMV reverse transcriptase 5U, template RNA 5μl, 1×ThermoPol Reaction Buffer.

[0068] 3. Reaction conditions...

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Abstract

The invention discloses a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and a detection method for grass carp reoviruses (GCRV), and belongs to the technical field of the detection of fish viruses. The RT-LAMP assay kit for the grass carp reoviruses comprises the following ingredients: 1*ThermoPol Reaction Buffer, Bacillus stearothermophilus deoxyribonucleic acid (BstDNA) polymerase, deoxyribonucleotide triphosphate (dNTPs), AMV reverse transcriptase, outer primers (F3 and B3), inner primers (FIP and BIP), Betaine, MgCl2, dithiothreitol (DTT) and 1,000*SYBR Green I. The assay kit has the characteristics of simpleness, convenience, quickness and high specificity and sensitivity; GCRV-104 in a sample can be accurately detected within 2 hours only by using a water bath kettle or metal bath; and the assay kit can detect grass carp tissues infected by the GCRV-104 and cells (such as cytokine-induced killer (CIK) cells) infected by the GCRV-104, and is applicable to the on-site rapid detection of the GCRV-104.

Description

technical field [0001] The invention belongs to the technical field of fish virus detection, in particular to a grass carp reovirus RT-LAMP detection kit, and also to a grass carp reovirus detection method. Background technique [0002] Grass carp haemorrhagic disease is a serious viral disease with strong infectivity and pathogenicity, which mainly harms grass carp fingerlings. It was first discovered in Shikou, Hubei in 1972, and named grass carp hemorrhagic disease. In 1978, it was first confirmed by electron microscopy that the pathogen was a virus. The pathogen of grass carp hemorrhagic disease outbreak was isolated for the first time in 1983, and the virus was identified as reovirus according to its morphological and physical and chemical characteristics. In 1991, the fifth meeting of the International Committee on Taxonomy of Viruses officially named the virus as Grass carp reovirus (GCRV), and included it in the genus Aquareovirus of the family Reoviridae. Grass c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 张辉曾令兵周勇范玉顶徐进张金风
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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