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One-step fluorescent quantitative RT-PCR detection primers and kits for Nodamura virus in Macrobrachium rosenbergii

A Noda Village virus and fluorescence quantitative technology, which is applied in the determination/testing of microorganisms, microorganisms, and methods based on microorganisms, etc., can solve the problems of low sensitivity, inability to quantify, and easy cross-contamination, and achieve sensitive and rapid operation, rapid detection, The effect of convenient operation

Inactive Publication Date: 2018-08-28
GUANGXI ACADEMY OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual detection work, in situ hybridization and ELISA detection takes a long time; although colloidal gold test strips are fast and easy to operate, but the sensitivity is low; RT-PCR is difficult to detect latent infection samples; nested RT -PCR and RT-LAMP are sensitive and fast, but they are prone to cross-contamination and cannot be quantified

Method used

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  • One-step fluorescent quantitative RT-PCR detection primers and kits for Nodamura virus in Macrobrachium rosenbergii
  • One-step fluorescent quantitative RT-PCR detection primers and kits for Nodamura virus in Macrobrachium rosenbergii
  • One-step fluorescent quantitative RT-PCR detection primers and kits for Nodamura virus in Macrobrachium rosenbergii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, primer design and synthesis

[0033] A pair of specific primers were designed according to the conserved region of the MrNV gene sequence (GenBank No. HQ287005). The size of the augmented fragment is 119bp, which was synthesized by Treasure Bioengineering (Dalian) Co., Ltd., diluted with DEPC-treated water to 20 pmol / μl, and stored at -20°C for later use.

Embodiment 2

[0034]Embodiment 2, the establishment of one-step fluorescent quantitative RT-PCR detection method

[0035] (1) Sample preparation

[0036] Take 50-100 mg of the gill filaments and muscle tissue of the juvenile or adult shrimp to be tested, add 500 μl of TN buffer (20 mM Tris / HCl, 0.4 M NaCl, pH 7.4) to homogenate, and centrifuge to obtain the supernatant. The RNA extraction kit (QIAamp Viral RNA MiniKit) was used to extract the total RNA of Macrobrachium rosenbergii Nodamura virus according to its instructions, and the purity and concentration of virus RNA were measured with a nucleic acid protein analyzer, and stored at -80°C for later use.

[0037] (2) Preparation of standard products

[0038] The MrNV capsid protein gene fragment was amplified using the previously constructed and preserved plasmid as a template, and cloned into the pGM-T vector. After the positive identification, the plasmid DNA was extracted and linearized by single enzyme digestion, and the recovered pr...

Embodiment 3

[0047] Embodiment 3, the assembly of detection kit

[0048] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use. The kit contains the following reagents:

[0049] (1) One-step fluorescence quantitative RT-PCR reaction solution (solution A): a total of 2.3mL, each dose of reaction solution is 23μL, composed of 2×OneStep RT-PCR Buffer 12.5 μL, PrimeScript 1 step Enzyme Mix 1.0 μL, upstream and downstream primers with a concentration of 10 μmol / L 1.0 μL each, ROX Reference Dye II (50X) 0.5 μL, H 2 O 7.0 μL composition.

[0050] (2) Positive control (solution P): RNA fragment containing MrNV gene, 200 μL;

[0051] (3) Negative control (N solution): RNA fragments without MrNV gene, 200 μL;

[0052] (4) Working standard: including 1×10 8 (B1 liquid), 1×10 7 (B2 liquid), 1×10 6 (B3 solution), 1×10 5 (B4 liquid), 1×10 4 (B5 liquid), 1×10 3 (B6 liquid), 1×10 2 (B7 liquid), 1×10 1 (B8 solution) 200 μL each copy / μL standard RNA. ...

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Abstract

The invention discloses a primer for one-step fluorescent quantitative RT-PCR detection of Nodamura virus of macrobrachium rosenbergii. The primer comprises a sense primer MrNV-QF1 and a downstream primer MrNV-QR1, wherein the sense primer MrNV-QF1 has a base sequence as shown in the sequence table SEQ.ID.No.1, and the downstream primer MrNV-QR1 has a base sequence as shown in the sequence table SEQ.ID.No.2, according to which, the inventor establishes the one-step fluorescent quantitative RT-PCR detection method for Nodamura virus of macrobrachium rosenbergii and prepares the corresponding detection kit. The detection method and the corresponding detection kit are applied to detection of MrNV, does not need to separate inverse transcription from PCR reaction, are convenient, rapid and sensitive to operate, and beneficial for rapid detection of MrNV and prevention and control of WTD.

Description

technical field [0001] The invention belongs to the field of molecular biology detection of viruses, in particular to a primer and a kit for detecting Macrobrachium rosenbergii Nodamura virus by one-step fluorescent quantitative RT-PCR. Background technique [0002] Macrobrachium rosenbergii nodavirus (MrNV) is an important pathogen of Macrobrachium rosenbergii white tail disease (WTD), which mainly harms desalinated M. Symptoms, a large number of deaths in a short period of time, the cumulative mortality rate in shrimp ponds is generally 30% to 70%, and when it is severe, it reaches more than 90%. WTD was first reported in Thailand by Nash et al. in 1987, and then gradually spread to Taiwan, France, Guateloupe and Antilles, and mainland China. In my country, WTD first appeared in Guangdong and Guangxi around 1996, and then quickly spread to Zhejiang, Jiangsu, Shanghai and other places. Since 2000, it has been widely popular throughout the country, which has greatly affecte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/70C12Q1/701C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 童桂香韦信贤黎小正吴祥庆杨琼李旻
Owner GUANGXI ACADEMY OF FISHERY SCI