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SsDNA aptamer for identifying micropterus salmoides iridovirus and application thereof

A nucleic acid aptamer and perch rainbow technology, which is applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of lack of rapid detection methods for LMBV and few research reports, and achieve high library enrichment efficiency and reproducibility. Reproducibility, high affinity and specificity

Active Publication Date: 2021-03-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A kind of ssDNA nucleic acid aptamer and its application for identifying largemouth bass iridescent virus provided by the present invention are mainly used to solve the current research on LMBV nucleic acid aptamer Few reports, lack of rapid detection methods for LMBV

Method used

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  • SsDNA aptamer for identifying micropterus salmoides iridovirus and application thereof
  • SsDNA aptamer for identifying micropterus salmoides iridovirus and application thereof
  • SsDNA aptamer for identifying micropterus salmoides iridovirus and application thereof

Examples

Experimental program
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Effect test

experiment example 1

[0085] The purpose of Experimental Example 1 is to measure and analyze the affinity constants of the three nucleic acid aptamers (LA38, LB49, LA13) obtained in the above examples and LMBV respectively. The ELASA method was used to analyze the binding affinity of the nucleic acid aptamer to LMBV, and the specific steps were as follows:

[0086] (1) Dilute the 5'-biotinylated nucleic acid aptamer to different concentrations (0-1000nM), denature at 95°C for 5 minutes, cool on ice for 10 minutes to denature, and then add to the enzyme coated with LMBV Combine in wells of linked plates for 1 hour at 4°C in the dark;

[0087] (2) Wash the corresponding wells with PBS buffer 5 times again, after the incubation, wash 3 times with PBS, add HRP-Strep (1:10000 dilution), and incubate for 30 minutes;

[0088] (3) After washing with PBS for 5 times, use the Pierce TMB substrate kit to develop color, and read the OD corresponding to the well 450 value;

[0089] (4) The mean value of the ...

experiment example 2

[0092] The purpose of Experimental Example 2 is to analyze the toxicity of the three nucleic acid aptamers (LA38, LB49, LA13) obtained in the above examples. Use the kit (CCK-8) to measure the toxicity of the nucleic acid aptamer at the cellular level, and the specific steps are as follows:

[0093] (1) Culture carp epithelial tumor cells (EPC) in a 96-well plate for 24 hours, discard the culture medium, add different concentrations (1-1000nM) of pre-denatured nucleic acid aptamers, and continue to culture at 28°C for 48 hours. Treated EPC cells were used as controls;

[0094] (2) According to the operation method of the kit manual, add 10 μL of CCK-8 solution to each well, and incubate at 28°C for 4 hours;

[0095] (3) Use a microplate reader to detect the absorbance at 450 nm.

[0096] Using the CCK-8 cell viability assay kit, the cell viability results of EPC cells after treatment with different concentrations of nucleic acid aptamers were as follows: Figure 5 As shown,...

experiment example 3

[0105] The purpose of Experimental Example 3 is to analyze the effects of the three nucleic acid aptamers (LA38, LB49, and LA13) obtained in the above-mentioned examples on in vitro resistance to LMBV infection. Specific steps are as follows:

[0106] The random starting library (Library) and the screened 3 nucleic acid aptamers (200nM) were added to the EPC cells respectively as 4 experimental groups, and the EPC cells without any treatment were used as the blank group (Mock for short), After 24 hours, observe the cell growth situation of each group, the results are as follows: Figure 7 As shown, it can be seen that the treatment of EPC cells with nucleic acid aptamers has no effect on cell growth.

[0107] In addition, the random starting library (Library) was incubated with LMBV at 4°C for 1 hour and then added to the EPC cells. As a control group, the three nucleic acid aptamers (200nM) screened were incubated with LMBV at 4°C After 1 hour, it was added to the EPC cells...

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Abstract

The invention relates to the technical field of aquatic product virus detection, and discloses an ssDNA aptamer for identifying micropterus salmoides iridovirus. The nucleotide sequence of the ssDNA aptamer is shown as SEQ ID NO: 1-3, and the aptamer has the advantages of high specificity, high affinity, zero cytotoxicity, easy modification and substitution of different parts of the aptamer, and easy preservation. A prediction secondary structure of the aptamer provided by the invention has a unique stem-loop structure, the structure is stable and the target binding capacity is strong, so thatthe aptamer is suitable for preparing a micropterus salmoides iridovirus detection probe and a drug carrier and is applied to separation and purification of drugs; and rapid and efficient identification detection or treatment of micropterus salmoides iridovirus is facilitated, and the aptamer has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of aquatic virus detection, and more specifically, to an ssDNA nucleic acid aptamer for identifying largemouth bass iridescent virus and its application. Background technique [0002] Largemouth bass is one of the important freshwater farmed fishes, and it is widely farmed all over the world because of its fast growth, high nutrition and delicious meat. Largemouth bass iridescent virus (LMBV) is a viral pathogen isolated from diseased largemouth bass, with a high fatality rate. LMBV is a nucleoplasmic double-stranded DNA virus belonging to the Ranavirus genus of the family Iridoviridae. Typical symptoms caused by LMBV infection in largemouth bass include lethargy, internal and external bleeding, and organ enlargement. In recent years, large-scale LMBV infection has occurred frequently in largemouth bass farming, causing huge economic losses and threatening the healthy development of largemouth bass farming...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10G01N33/569
CPCC12N15/115C12N15/1048G01N33/56983C12N2310/16Y02A40/81
Inventor 秦启伟张欣悦王劭雯黄晓红刘嘉昕王立群张泽妙
Owner SOUTH CHINA AGRI UNIV
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