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Rapid detection primer set and kit for macrobrachium rosenbergii movement disorder nodaviruses

A technology of Nodamura virus and Macrobrachium rosenbergii, applied in the field of rapid detection of Nodamura virus on-site primer sets and kits, which can solve the problem of inability to detect and warn "iron shrimp" pathogens at an early stage, and achieve the realization procedure Standardization and standardization, improved detection level, good specificity

Pending Publication Date: 2020-12-22
江苏数丰水产种业有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a fast and sensitive detection method for Macrobrachium rosenbergii action obstacle Nodamura virus suitable for use on the production site, and standardize the concentration of detection reagents in a unified matching kit to overcome the inability in the current industry The lack of early detection and early warning of the "iron shrimp" pathogen makes the detection of MMDNV more convenient, rapid and sensitive

Method used

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  • Rapid detection primer set and kit for macrobrachium rosenbergii movement disorder nodaviruses
  • Rapid detection primer set and kit for macrobrachium rosenbergii movement disorder nodaviruses
  • Rapid detection primer set and kit for macrobrachium rosenbergii movement disorder nodaviruses

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Embodiment 1

[0037] Example 1 Design and screening of primers for on-site rapid detection of Macrobrachium rosenbergii mobility disorder Nodamura virus

[0038]First, the RNA-dependent RNA polymerase (RdRp) gene of Macrobrachium rosenbergii MMDNV cloned from Jiangsu, Zhejiang, Guangdong, Guangxi and other places suffering from iron shrimp disease was compared with the NCBI online program Blastn and the molecular biology software BioEitd7.0. For the variation of the above sequences, the conserved sequence of the RdRp gene of MMDNV was selected to design amplification primers through the software LampDesigner 4.0, and a total of 3 sets of primers were designed (Table 1).

[0039] Table 1: Amplification primers designed based on MMDNV protein A gene

[0040]

[0041]

[0042] Using the three sets of primer combinations designed and synthesized above, the reaction system was prepared according to the following components and the amplification reaction was carried out (25 μL / reaction, ref...

Embodiment 2

[0045] Embodiment 2 Detection kit of the present invention is made up of following parts (can detect the packing of 8 samples):

[0046] (1) Sample collection tubes, 8 pieces, used to hold and grind samples to be tested;

[0047] (2) Nucleic acid washing tubes, 10 pieces, each tube is filled with 1mL double distilled water;

[0048] (3) Template denaturation tubes, 10, each filled with 20 μL of TE buffer (containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0);

[0049] (4) Amplification chromogenic tubes, 10 pieces, a single tube contains 24 μL of amplification reaction solution and 1 μL of dye, the composition of the amplification reaction solution is as follows: each 1.6 μM of the upstream primer 1 and downstream primer 1 of the amplification primers, the amplification The melting primer 1 and 2 of the primers are 0.8 μM, the upstream primer 2 of the amplification primer is 0.2 μM, each of dATP, dTTP, dGTP and dCTP is 1.4 mM, MgCl 2 2mM, Betaine (betaine) 1.2M, Tris-HCl 20mM, K...

Embodiment 3

[0055] Embodiment 3 The detection kit of the present invention can also be made up of following parts (can detect the packing of 8 samples):

[0056] (1) Sample collection tubes, 8 pieces, used to hold and grind samples to be tested;

[0057] (2) Nucleic acid washing tubes, 10 pieces, each tube is filled with 1mL double distilled water;

[0058] (3) Template denaturation tubes, 10, each containing 20 μL of TE buffer (containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0);

[0059] (4) Amplification chromogenic tubes, 10, each tube is equipped with 25 μL of amplification reaction solution and 1 μL of nucleic acid dye, the components of the amplification reaction solution are as follows: each of the upstream primer 1 and downstream primer 1 of the amplification primer is 1.6 μM , 10.8 μM melting primer of amplification primer, 0.2 μM each of upstream primer 2 and downstream primer 2 of amplification primer, 1.4 mM each of dATP, dTTP, dGTP and dCTP, MgCl 2 8mM, Betaine (betaine) 1.2M...

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Abstract

The invention relates to a rapid detection primer set and kit for macrobrachium rosenbergii movement disorder nodaviruses (MMDNV). The primer set comprises five primers. The kit comprises sample collection tubes, nucleic acid washing tubes filled with double distilled water, template denaturation tubes filled with a TE buffer solution, amplification chromogenic tubes filled with an amplification reaction solution and a dye, a negative control tube, a positive control tube and FTA membranes. The primer set provided by the invention can specifically detect the target nucleic acid of the low-copyMMDNV, and a kit detection method provided by the invention has higher convenience, sensitivity and specificity than a conventional PCR detection method, a conventional histopathology detection method, an conventional electron microscope detection method and the like, and is convenient to use, more accurate and quicker in detection and low in use cost. The primer set and the kit disclosed by theinvention can be used in a conventional detection laboratory and can also be used in a field production field.

Description

technical field [0001] The invention belongs to the technical field of detection of marine biological pathogens, and in particular relates to a primer set and a kit for on-site rapid detection of Macrobrachium rosenbergii movement disorder nodavirus (MMDNV for short). Background technique [0002] In recent years, the Macrobrachium rosenbergii industry in my country has encountered a series of problems, among which the serious disease of iron shrimp is more prominent. The yield per mu of normal Macrobrachium rosenbergii is 300-400 kg, but the yield per mu of shrimp ponds with "iron shrimp" is only 100-200 kg, and the adults of Macrobrachium rosenbergii are small and light in weight during the harvest period, and the price is also higher than that of normal commodities. The size of shrimp is much lower, which seriously affects the income of farmers. Farmers who encounter "iron shrimp" are basically in a state of loss. In 2014, there were mass incidents caused by thousands of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 张庆利杨国梁黄倢董宣万厚荣夏正龙
Owner 江苏数丰水产种业有限公司