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RT-LAMP detection primer group, kit and method for tilapia lake virus

A technology of RT-LAMP and kits, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., and can solve the problem of high false positive rate of nested and semi-nested RT-PCR and the operation process Complicated, time-consuming and other issues, to achieve the effect of easy reaction results, simple reaction conditions, and simple operation

Active Publication Date: 2018-05-11
PEARL RIVER FISHERY 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 detection methods for TiLV include nested and semi-nested RT-PCR, and in situ hybridization. The operation of these detection methods is relatively complicated, time-consuming, and requires specific equipment. Therefore, these existing detection methods are not suitable for efficient and rapid on-site detection of Luohu virus

Method used

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  • RT-LAMP detection primer group, kit and method for tilapia lake virus
  • RT-LAMP detection primer group, kit and method for tilapia lake virus
  • RT-LAMP detection primer group, kit and method for tilapia lake virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 detects the RT-LAMP primer set of Luohu virus

[0043] After screening a large number of primers designed by this application, it was found that the primer sets TiLV-FIP, TiLV-BIP, TiLV-F3, TiLV-LB, TiLV-B3, and TiLV-LF have the effect on the detection of Luohu virus by RT-LAMP method Preferably, its base sequence is as follows.

[0044] Outer primers:

[0045] TiLV-FIP: 5'-CTTCTGCGAGTCTTTGAGGCACCTGATGGCCCAGACACTA-3' (SEQ ID NO: 1);

[0046] TiLV-BIP:5'-AGAACCCGACTAGAGGCTCTCTCTGACACGAGGAGCCTATG-3' (SEQ ID NO: 2);

[0047] Inner primer:

[0048] TiLV-F3: 5'-CTGACTTGGGATTGCCACC-3 (SEQ ID NO: 3);

[0049] TiLV-B3: 5'-TGCCTAGCTGCTCTGATCT-3 (SEQ ID NO: 4);

[0050] Loop primer:

[0051] TiLV-LF: 5'-AGGACAGACAGGACTTTCTGATC-3 (SEQ ID NO: 5);

[0052] TiLV-LB: 5'-CAGACTTGGACCCCCTGGT-3 (SEQ ID NO: 6).

Embodiment 2

[0053] Embodiment 2 detects the RT-LAMP kit of Luohu virus

[0054] The kit includes the following components:

[0055] (1) The RT-LAMP primer set designed in Example 1.

[0056] (2) RT-LAMP reaction solution: containing 23-2.7mM dNTPs, 14-18mM MgSO 4 , 1.4~1.8mM betaine, 18~22mM KCl, 0.15~0.25% Tween20, 18~22mM (HN 4 ) 2 SO 4 , 35-45mM Tris-Cl pH 8.6-9.0.

[0057] (3) Bst DNA polymerase.

[0058] (4) AMV reverse transcriptase.

[0059] (5) Fluorescent dye: calcein.

[0060] (6) Positive and negative control samples: the positive control is plasmid DNA containing the target gene fragment, and the negative control is DEPC water.

Embodiment 3

[0061] Embodiment 3 detects the method for Luohu virus RT-LAMP

[0062] ① Extraction of viral RNA

[0063] According to the instructions of the Trizol kit (purchased from Invitrogen, product number: 15596-026) or according to the instructions of the Qiagen RNA extraction kit (purchased from Qiagen, product number: 74104), RNA was extracted from the internal organs of fish infected with the corresponding virus. , as a template for RT-PCR reactions. The main steps of Trizol extraction of total RNA are as follows: take the tissue sample and add 500 μl Trizol reagent, mix well and let it stand for 5 minutes, add 400 μl chloroform, shake and mix for 1 minute, centrifuge at 12000 rpm at 4°C for 15 minutes, take the supernatant and add an equal volume of isopropyl Alcohol, mix upside down, place at 4°C for 15min, centrifuge at 12,000rpm at 4°C for 15min, discard the supernatant, wash the pellet with 70% ethanol, centrifuge at 12,000rpm at 4°C for 5min, and resuspend the pellet with ...

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Abstract

The invention discloses a RT-LAMP detection primer group, a kit and a method for a tilapia lake virus. The kit comprises RT-LAMP reaction liquid, Bst DNA polymerase, AMV reverse transcriptase, fluorescent dye, the RT-LAMP primer group, distilled water and positive-and-negative control samples; the RT-LAMP primer group is composed of a pair of outer primers, a pair of inner primers and a pair of loop primers. The RT-LAMP detection kit for the tilapia lake virus has the advantages of being easy and convenient to operate, high in specificity and sensitivity, rapid, accurate and the like, is quitesuitable for export quarantine and site detection of farms, and is easily applied and popularized in a large scale.

Description

technical field [0001] The invention belongs to the field of virus molecular biology, in particular to an RT-LAMP visual rapid detection kit for Luohu virus, more specifically to an RT-LAMP primer set, detection method and visual rapid detection kit for Luohu virus. Background technique [0002] Since 2000, my country's tilapia aquaculture production has been ranked first in the world. In recent years, my country's tilapia aquaculture production has maintained a growth trend. In 2015, the total production of tilapia was about 1.78 million tons, accounting for about 30% of the global total production. As an export-oriented industry in my country, the tilapia industry mainly relies on export trade to maintain the operation of the industry. There are 133 tilapia processing and export enterprises in China, with a total export volume of about 400,000 tons, sold to 97 countries and regions. Since 2002, the export volume and export value of tilapia in my country have basically sho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 曾伟伟王庆王英英尹纪元汤亚方石存斌张德峰李莹莹任燕常藕勤
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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