Reagent and method for visual lamp detection of prawn taura syndrome virus

A technology of syndrome virus and detection reagent, applied in the field of warm amplification detection, to achieve the effect of improved sensitivity, simple instrument and strong specificity

Active Publication Date: 2016-03-02
GUANGXI VETERINARY RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the detection of prawn taura syndrome virus (TSV) by loop-mediated isothermal amplification (LAMP).

Method used

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  • Reagent and method for visual lamp detection of prawn taura syndrome virus
  • Reagent and method for visual lamp detection of prawn taura syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: specificity test

[0042] Step (1) Sample RNA extraction: Take about 100 mg of the hepatopancreas, gills, and muscle tissue of the prawns for tissue homogenization, and then take an appropriate amount of samples, use Trizol RNA extraction reagent (GIBCOBRL, USA) to extract the total RNA of the prawns, and place in Store at -20°C for later use.

[0043] Step (2) Preparation of LAMP detection liquid system

[0044] ①: For the capsid protein gene sequence in the conserved region of the shrimp taura syndrome virus genome, design 6 specific amplification primers, including two outer primers F3 and B3, two inner primers FIP and BIP, and two loop primers LF and LB.

[0045] F3 is 5'-GGTCCTGGACTTCAAACA-3';

[0046] B3 is 5'-TTGCGTGGTGGGACTA-3';

[0047] FIP is 5'-AGGTTCCACAATCTTGATGAATGAT-CTGTTACATTCTTAGACAGCACTA-3';

[0048] BIP is 5'-TCACTGTTAGTAACACTACCTCCT-CCTGCTAACCCAGTCGA-3';

[0049] LF is 5'-ATCCCAATCACTAATCAGAATG-3';

[0050] LB is 5'-AGTCCTCCACT...

Embodiment 2

[0061] Embodiment 2: specificity test

[0062] The difference between this example and Example 1 lies in the various reagents required in the preparation of the LAMP detection liquid system in step (2) LAMP detection liquid system for shrimp taura syndrome virus, and the addition amounts and concentrations of the reagents are as follows:

[0063]0.5 μL 10 μmol / L F3 (final concentration 0.2 μmol / L), 0.5 μL 10 μmol / L B3 (final concentration 0.2 μmol / L), 2.0 μL 100 μmol / L FIP (final concentration 8 μmol / L), 2.0 μL 100 μmol / L BIP (final concentration 8 μmol / L), 0.5 μL 10 μmol / LLF (final concentration 0.2 μmol / L), 0.5 μL 10 μmol / L LB (final concentration 0.2 μmol / L), 2.5 μL 10× reaction buffer BstDNA polymerase buffer (final concentration 1×), 2.5 μL 40 U / μL strand-displacing DNA polymerase (final concentration 4 U), 2.5 μL 100 mmol / L MgSO 4 (final concentration 10mmol / L), 0.5μL 1mmol / L MnCl 2 (final concentration 0.02mmol / L), 1μL 25mmol / L betaine (final concentration 1.0mmol / L...

Embodiment 3

[0066] Embodiment 3: specificity test

[0067] The difference between this example and Example 1 lies in the various reagents required in the preparation of the LAMP detection liquid system in step (2) LAMP detection liquid system for shrimp taura syndrome virus, and the addition amounts and concentrations of the reagents are as follows:

[0068] 1.0 μL 200 μmol / L F3 (final concentration 8 μmol / L), 1.0 μL 200 μmol / L B3 (final concentration 8 μmol / L), 2.0 μL 2.5 μmol / L FIP (final concentration 0.2 μmol / L), 2.0 μL 2.5 μmol / L L of BIP (final concentration 0.2 μmol / L), 1.0 μL 200 μmol / LLF (final concentration 8 μmol / L), 1.0 μL 200 μmol / L LB (final concentration 8 μmol / L), 2.5 μL 10× reaction buffer BstDNA polymerase buffer (final concentration 1× ), 2.5 μL 40U / μL strand displacement DNA polymerase (final concentration 4U), 1.0 μL 20mmol / L MgSO 4 (final concentration 0.8mmol / L), 1.25μL of 25mmol / L MnCl 2 (final concentration 1.25mmol / L), 1μL 1mmol / L betaine (final concentration...

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Abstract

The invention relates to a visualization loop-mediated isothermal amplification detection reagent and a detection method of prawn taura syndrome virus. For the capsid protein gene sequence of the conserved region of the prawn taura syndrome virus genome, 6 specific amplification primers are designed, Amplification primers and reaction reagents together constitute the LAMP detection solution system, and the reaction reagents include reaction buffer Bst? DNA? polymerase? bufferr, strand displacement DNA polymerase, magnesium ion, divalent manganese ion, betaine, deoxyribonucleoside triphosphate dNTPs, color indicator, and the balance is water. The detection steps include: extracting the genomic DNA of the sample to be tested, preparing a LAMP detection solution system, a reaction system, an amplification reaction in a constant temperature device, and judging the detection result. On the premise of high sensitivity and strong specificity, the detection method of the invention uses simple instruments and low cost.

Description

technical field [0001] The invention belongs to the technical field of loop-mediated isothermal amplification detection, and in particular relates to a visual loop-mediated isothermal amplification (LAMP) detection reagent and a detection method of prawn taura syndrome virus. Background technique [0002] Shrimp Taura Syndrome Virus (TSV) is a newly discovered virus that seriously harms cultured shrimp in recent years. According to the 7th report of the International Committee on Taxonomy of Viruses (ICTV), the virus belongs to the Dicistroviridae family. Bee paralysis virus (Aparavirus). The virus mainly infects Pacific white shrimp (Pacific whiteshrimp), vannamei white shrimp (Penaaeus vannamei), Pacific blue shrimp (Pacific blueshrimp) and so on. [0003] Due to the serious harm of TSV, it has become one of the main diseases threatening the shrimp farming industry. Therefore, in 1995, it was listed as a must by the International Office of Epizootics (OIE), the United Foo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 谢芝勋庞耀珊谢丽基谢志勤邓显文刘加波范晴罗思思
Owner GUANGXI VETERINARY RES INST
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