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Method for detecting small RNA (Ribonucleic Acid) viruses of freshwater long-arm prawns

A technology for freshwater long-arm prawns and RNA viruses, which is applied in the detection field of aquatic viruses, can solve problems such as short time consumption, and achieve the effects of strong specificity, good linear relationship and high sensitivity

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

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Problems solved by technology

The fluorescent quantitative PCR method takes less time, and by comparing with the absolute quantitative standard curve, the sample can be quantitatively detected, and it has also been widely used in the detection of aquatic diseases, such as the infectious subcutaneous infection of Litopenaeus vannamei constructed by Liu Baobin et al. Fluorescent quantitative PCR detection method of Hematopoietic Necrosis Virus and Shrimp Enteroplasma Hepatica Fluorescent quantitative PCR detection of (EHP)[J]. Fishery Science Advances, 2017,38(002):158-166.), but there is no PCR detection method for MrPV-7 at present

Method used

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  • Method for detecting small RNA (Ribonucleic Acid) viruses of freshwater long-arm prawns
  • Method for detecting small RNA (Ribonucleic Acid) viruses of freshwater long-arm prawns
  • Method for detecting small RNA (Ribonucleic Acid) viruses of freshwater long-arm prawns

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Experimental program
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Effect test

Embodiment 1

[0065] The design of embodiment 1 nested PCR primer and the establishment of PCR method

[0066] 1. Design of nested PCR primers

[0067] The present invention discovers a new small RNA virus tentatively named Macrobrachium rosenbergii picornavirus 7 (MrPV-7) by enriching the viruses in freshwater long-armed prawns exhibiting "iron shell" symptoms and sequencing the macrovirome. ), whose genome sequence is shown in SEQ ID NO.1.

[0068] The present invention refers to the phylogenetic analysis of the Picornaviridae family Picornaviridae by the International Committee on Taxonomy of Viruses (ICTV), and through Bayesian tree building, the representative species of multiple genera in 8 families under Picornavirales are not classified into The tentative species of the genus, as well as multiple MrPV viruses (MrPV-1, MrPV-7, MrPV-4 and MrPV-12) discovered by the inventors of the present invention were analyzed phylogenetically. Because the RNA polymerase of Picornaviridae is resp...

Embodiment 2

[0089] Example 2 Optimization of nested PCR reaction conditions and detection of sensitivity and specificity

[0090] 1. Optimization of annealing temperature

[0091] For the convenience of comparing the impact of different annealing temperatures on nested PCR amplification, the present invention uses a lower concentration of (10 4 copies / uL) of the positive recombinant plasmid as a template, the first round of nested PCR amplification primer MrPV-7-1-F / R and the second round of amplification primer MrPV-7-2- F / R (hereinafter referred to as the first expansion and the second expansion) is amplified, and the annealing temperature corresponding to the brightest band is selected as the optimal annealing temperature. The reaction system is the same as in Example 1, and the experiment is repeated 3 times.

[0092] The results of the first round of nested PCR primers MrPV-7-1-F / R are as follows Figure 4 As shown, the annealing temperatures corresponding to lanes 1 to 6 are 50°C,...

Embodiment 3

[0106] The nested PCR detection of embodiment 3 actual samples

[0107] The present invention utilizes the Nest's PCR detection method constructed to detect the gill samples of 23 freshwater long-armed prawns collected from Huzhou City, Zhejiang Province, and the detection results of the first expansion and the second expansion primers are respectively as follows: Figure 10 and Figure 11 As shown, no bands appeared in the first round of nested PCR amplification, and 17 samples were positive results in the second round of amplification, with a positive detection rate of 73.9%.

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Abstract

The invention discloses a method for detecting small RNA (Ribonucleic Acid) viruses of freshwater long-arm prawns, namely a nested PCR (Polymerase Chain Reaction) and fluorescent quantitative PCR detection method of MrPV-7, which can be used for screening seed prawns or prawn seeds and the like without viruses so as to reduce the loss of the freshwater long-arm prawns in a culture process. The nested PCR and fluorescent quantitative PCR method for detecting the small RNA viruses of the freshwater long-arm prawns, established by the invention, is high in sensitivity and good in specificity and repeatability, and besides qualitative detection of the viruses, the nested PCR and fluorescent quantitative PCR method also can be used for quantitative detection.

Description

technical field [0001] The invention belongs to the technical field of aquatic virus detection. More specifically, it relates to a method for detecting small RNA viruses of freshwater longarm prawns. Background technique [0002] The freshwater long-armed prawn (Macrobrachium rosenbergii) has been introduced into my country as a foreign species for more than 40 years. Because of its remarkable economic benefits, it has become the main freshwater cultured shrimp in my country. However, the occurrence of diseases has affected the development of aquaculture industry. Viruses currently reported in China that can infect freshwater longarm prawns and cause diseases include: white spot syndrome virus (white spot syndrome virus, WSSV), Noda village virus (Macrobrachium Rosenbergii Nodavirus, MrNV) and parvo-like virus (parvo-like virus). , HPV), etc., there are still some diseases that have not yet identified the pathogen. With the in-depth research on aquatic diseases, more and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6848C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848C12Q1/6851C12Q2531/113C12Q2549/119C12Q2565/125C12Q2563/107C12Q2545/114
Inventor 何建国缪琪瑾翁少萍周丹丹
Owner SUN YAT SEN UNIV
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