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Nucleic acid aptamer and application thereof in detection of cells infected by micropterus salmoides iridovirus

A nucleic acid aptamer and virus infection technology, which is applied in the directions of measurement devices, biochemical equipment and methods, instruments, etc., can solve the problems of cell detection that cannot be infected by largemouth bass iridovirus, and achieves convenient in vitro chemical synthesis and small molecular weight. , the effect of short preparation cycle

Pending Publication Date: 2021-08-06
GUANGXI ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention is to provide a high-specificity, high-sensitivity, non-immunogenic, stable and easy-to-modify nucleic acid aptamer for detecting cells infected by largemouth bass iridescent virus, which is convenient for synthesis and storage, to at least solve the existing Problems with the inability of current biological assays to rapidly and accurately detect largemouth bass iridescent virus-infected cells in the field

Method used

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  • Nucleic acid aptamer and application thereof in detection of cells infected by micropterus salmoides iridovirus
  • Nucleic acid aptamer and application thereof in detection of cells infected by micropterus salmoides iridovirus
  • Nucleic acid aptamer and application thereof in detection of cells infected by micropterus salmoides iridovirus

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Embodiment 1

[0020] Embodiment 1: the screening of nucleic acid aptamer

[0021] Step 1: Synthesize a random ssDNA library and primers shown in the sequence below

[0022] Random starting single-stranded ssDNA library Library:

[0023] 5'-GACGCTTACTCAGGTGTGACTCG-N50-CGAAGGACGCAGAGAAGTCTC-3'

[0024]5' primer: 5'-FAM-GACGCTTACTCAGGTGTGACTCG-3';

[0025] 3' primer: 5'-Biotin-GAGACTTCATCTGCGTCCTTCG-3;

[0026] Step 2: SELEX screening to obtain nucleic acid aptamers that specifically recognize the aquatic pathogenic bacteria largemouth bass iridescent virus

[0027] 2.1 Dissolve 10 nmol of the above random library in 500 μl PBS, bathe in constant temperature water at 92°C for 5 minutes, then quickly insert it into ice, and bathe in ice for 10 minutes, and incubate the treated random library with largemouth bass iridovirus LMBV-infected cells on ice for 40 minutes;

[0028] 2.2 After the incubation and binding are completed, remove the supernatant by centrifugation, wash the largemouth bass...

Embodiment 2

[0046] In the embodiment of the present invention, the nucleic acid aptamer of SEQ ID NO: 1 or SEQ ID NO: 2 obtained in Example 1 was used to assemble a fluorescent molecular probe (Aptamer LBVA1-Based Fluorescent Molecular Probe, LBVA1-AFMP) kit to form a A LBVA1-AFMP detection kit for largemouth bass iridovirus-infected cells. The molecular probe contains the aforementioned nucleic acid aptamer of SEQ ID NO: 1 or SEQ ID NO: 2.

[0047] The nucleic acid aptamers screened by the SELEX technology in the embodiments of the present invention have good affinity and specificity, and the nucleic acid aptamers in the embodiments of the present invention are stable in structure, and still have good aptamers after group labeling and modification. Affinity and specificity can be applied to fluorescent molecular probe detection kits, and the nucleic acid aptamer of the embodiment of the present invention has the characteristics of short preparation cycle, good reproducibility, and small ...

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Abstract

The invention discloses a nucleic acid aptamer and an application thereof in detection of cells infected by a micropterus salmoides iridovirus. The nucleotide sequence of the ssDNA nucleic acid aptamer is 5'-CACGGGGGGGATCGATATTGACTTGGTTCTGACTCACACCGTTACCTCTT-3'(SEQ ID NO:1) or 5'-GACGCTTACTCAGGTGTGACTCGCACGGGGGGGATCGATATTGACTTGGTTCTGACTCACACCGTTACCTCTTCGAAGGACGCAGATGAAGTCTC-3'(SEQ ID NO:2). The nucleic acid aptamer has specificity and high sensitivity on cells infected by the micropterus salmoides iridovirus, and does not have immunogenicity. The nucleic acid aptamer is stable in structure, easy to modify and convenient to synthesize and preserve, and can be used for rapidly and accurately detecting the cells infected by the micropterus salmoides iridovirus.

Description

technical field [0001] The present invention relates to a nucleic acid aptamer and its application, in particular to a nucleic acid aptamer and its application in the diagnosis, identification and detection of cells infected with largemouth bass iridescent virus. Background technique [0002] Because of its fast growth and delicious meat, largemouth bass has become an important species of freshwater aquaculture in my country, with an annual output of 470,000 tons. In recent years, with the increase of largemouth bass breeding density and the expansion of breeding scale, the problem of breeding diseases has become increasingly serious, causing huge economic losses. In 2019, fish diseases broke out in largemouth bass cultured in ponds in Nanning, Baise, Liuzhou, Hezhou and other places in Guangxi. The main symptoms of diseased fish were black body color, large ulcers on the body surface, muscle necrosis and bleeding, pectoral fins, caudal fins and The base of the dorsal fin i...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/569G01N21/64
CPCC12N15/115G01N33/56983G01N33/56966G01N21/6428C12N2310/16Y02A50/30
Inventor 李鹏飞余庆刘明珠苏美珍卓晓菲王浩王高学
Owner GUANGXI ACAD OF SCI
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