Small RNA (Ribonucleic Acid) virus gene of white foot shrimp and application of small RNA virus gene

A technology of RNA virus and white-legged shrimp, which is applied in the field of detection of aquatic pathogens, can solve problems such as production decline and loss of farmers, and achieve high sensitivity, good linear relationship, good repeatability and specificity

Pending Publication Date: 2022-04-19
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The appearance of "iron shell shrimp" in the breeding process has affected the development of the white-legged shrimp farming industry. Young white-leg shrimp infected with the "iron shell shrimp" disease will stop growing when they grow to 5cm to 6cm. It will not die, but the production will drop, causing huge losses to the farmers

Method used

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  • Small RNA (Ribonucleic Acid) virus gene of white foot shrimp and application of small RNA virus gene
  • Small RNA (Ribonucleic Acid) virus gene of white foot shrimp and application of small RNA virus gene
  • Small RNA (Ribonucleic Acid) virus gene of white foot shrimp and application of small RNA virus gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The design of embodiment 1 nested PCR primer and the establishment of method

[0060] 1. Design of nested PCR primers

[0061] The present invention has discovered a new small RNA virus, tentatively named Macrobrachium rosenbergii picorna virus12 (MrPV-12 ), whose genome sequence is shown in SEQ ID NO.1.

[0062] At the same time, the present invention refers to the phylogenetic analysis of Picornaviridae of Picornaviridae by the International Committee on Taxonomy of Viruses, selects a relatively conservative RNA polymerase gene, and constructs a tree through Bayesian, to represent representatives of multiple genera in 8 families of Picornavirales. species, tentative species not classified into genus, and a plurality of MrPV viruses (MrPV-1, MrPV-4, MrPV-7 and MrPV-12) found by the inventors of the present invention have carried out phylogenetic analysis, and the analysis results are as follows figure 1 shown. It can be seen from the figure that MrPV-12 is closely r...

Embodiment 2

[0078] The construction of embodiment 2 positive recombinant vector and the optimization of nested PCR reaction conditions

[0079] 1. Construction of positive recombinant vector pMD19-T-MrPV-12

[0080] Using the cDNA obtained by reverse transcription in Example 1 as a template, the method of the first round of nested PCR in Example 1 was used to amplify the gene sequence of SEQ ID NO.2 with a primer MrPV-12-1-F / R and sequenced . Compare the sequence obtained by sequencing with the assembled sequence of the macrovirus group. If the sequence is consistent, use the pMD of TaKaRa TM 19-T Vector Cloning Kit, the gel-cut recovery product was connected to the pMD-19T vector to construct a recombinant vector. carrier carrier carrier

[0081] 2. Optimization of annealing temperature

[0082] With a concentration of 10 4The positive recombinant vector of copies / uL is used as a template, and the first expansion primer MrPV-12-1-F / R and the second expansion primer MrPV-12-2-F / R ar...

Embodiment 3

[0090] Embodiment 3 Sensitivity, repeatability and specificity detection

[0091] 1. Sensitivity and repeatability detection

[0092] In order to detect the sensitivity and repeatability of the nested PCR primers, a concentration of 10 7 Copies / uL of positive recombinant vectors were sequentially diluted 10 times in 8 gradients. Using different concentrations of positive recombinant vectors as templates, PCR amplification was carried out with first-amplification and second-amplification primers respectively. The experiment was repeated three times, and the PCR products were detected by electrophoresis.

[0093] The sensitivity and repeatability detection results of the first amplification primers are as follows: Image 6 As shown, it can be seen from the figure that the results of the three amplifications are consistent, and the minimum detection limit of the primers for the first amplification is 10 5 copies / uL. Sensitivity and repeatability detection results of the secon...

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Abstract

The invention discloses a gene of a small RNA (Ribonucleic Acid) virus of white foot shrimps and application of the gene. On the basis of the small RNA virus gene of the white foot shrimp, the nested and fluorescent quantitative PCR detection method of the small RNA virus of the white foot shrimp is established. The established nested PCR method and fluorescent quantitative PCR method are high in sensitivity and good in repeatability and specificity, the lowest detection limit is 10 copies / microliter, and the nested PCR method and the fluorescent quantitative PCR method can be used for screening non-toxic seed shrimps or shrimp seeds and are suitable for being popularized and used in base-level culture units of freshwater shrimps.

Description

technical field [0001] The invention belongs to the technical field of detection of aquatic pathogens. More specifically, it relates to a white-legged shrimp picorna virus gene and its application. Background technique [0002] Macrobrachium rosenbergii (Macrobrachium rosenbergii), also known as Macrobrachium rosenbergii, is currently farmed in more than 10 provinces, municipalities, and autonomous regions, including Guangdong, Guangxi, Hunan, Hubei, Jiangsu, Shanghai, and Zhejiang, and has made great contributions to increasing farmers' income in many regions. important contribution. The appearance of "iron shell shrimp" in the breeding process has affected the development of the white-legged shrimp farming industry. Young white-leg shrimp infected with the "iron shell shrimp" disease will stop growing when they grow to 5cm to 6cm. It will not die, but the production will drop, which will bring huge losses to the farmers. [0003] White spot syndrome virus (white spot sy...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/11C12Q1/70C12Q1/6848C12Q1/6851
CPCC07K14/005C12Q1/701C12Q1/6848C12Q1/6851C12N2770/32022C12Q2521/107C12Q2549/119C12Q2531/113C12Q2563/107
Inventor 何建国缪琪瑾翁少萍周丹丹
Owner SUN YAT SEN UNIV
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