Oligonucleotide aptamer capable of specifically recognizing largemouth bass virus as well as screening method and application of oligonucleotide aptamer

A largemouth bass virus and oligonucleotide technology, which is applied in the field of oligonucleotide aptamers and its screening, can solve the problems of high cost, long time required for antibody preparation, instability, etc., and achieve low cost and high screening efficiency. The effect of short cycle time and easy operation

Active Publication Date: 2021-03-19
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these molecular diagnostic techniques have certain sensitivity and specificity, and can be detected in large quantities and avoid subjective bias, which partially solve the demand for rapid detection, the test process requires professional technicians and PCR gene amplification. Immunological methods have the advantages of high sensitivity, strong specificity, and easy observation, but antibody preparation requires a long time, high cost, and instability

Method used

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  • Oligonucleotide aptamer capable of specifically recognizing largemouth bass virus as well as screening method and application of oligonucleotide aptamer
  • Oligonucleotide aptamer capable of specifically recognizing largemouth bass virus as well as screening method and application of oligonucleotide aptamer
  • Oligonucleotide aptamer capable of specifically recognizing largemouth bass virus as well as screening method and application of oligonucleotide aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The screening method of the nucleic acid aptamer that specifically recognizes largemouth bass virus comprises the following steps:

[0045] 1) Synthesis of random single-stranded DNA (ssDNA) library and primers

[0046] A random ssDNA library of single-stranded DNA with a length of 81nt was established: 5′-AGTATACGTATTACCTGCAGC(N 40)CGATATCTCGGAGATCTTGC-3′, with fixed sequences at both ends and a random sequence with 40 bases in the middle N40.

[0047] Upstream primer F: 5'-AGTATACGTATTACCTGCAGC-3';

[0048]Downstream primer R: 5′-GCAAGATCTCCGAGATATCG-3′; the random ssDNA library and primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the random ssDNA library and primers were used ddH 2 O was prepared as a stock solution with a concentration of 10 μM and stored at -20°C for future use.

[0049] 2) Preparation of largemouth bass virus particles

[0050] After rinsing 50 mg of largemouth bass virus-positive liver with 0.0067M PBS buffer (pH7.4) f...

Embodiment 2

[0103] Eight diseased fish samples of largemouth bass were collected from different aquaculture farms in Zhejiang Province. The sample showed clinical signs of ulceration, exfoliation, superficial bleeding, and abdominal wall bleeding. Take 20 mg of liver from 8 samples of largemouth bass diseased fish, freeze-thaw and grind repeatedly, resuspend in 500 μL PBS, centrifuge at 8000 rpm for 5 min, and take the supernatant. Mix 50 μL of 40 nM LVA-3 nucleic acid aptamer aqueous solution with 50 μL of the above 8 supernatant samples, and incubate on a shaker at 25 °C and 100 rpm for 120 min; then add 50 μL of 15 mg / mL GO to the mixture and continue to incubate in the dark for 30 min , after centrifugation, the supernatant was taken for fluorescent quantitative PCR (the steps of fluorescent quantitative PCR here are the same as step 7 of the above-mentioned embodiment 1), and the Ct value was measured, thereby obtaining the concentration of aptamer bound to the target. The experimen...

Embodiment 3

[0105] 6 largemouth bass purchased randomly from the market were detected according to the method described in Example 2. Test results such as Figure 6 Shown: Sample 2 is LMBV positive, other samples are LMBV negative.

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Abstract

The invention discloses an oligonucleotide aptamer capable of specifically recognizing largemouth bass virus as well as a screening method and application of the oligonucleotide aptamer. By taking thelargemouth bass virus as a target, the oligonucleotide aptamer capable of being combined with the largemouth bass virus in a high-affinity and high-specificity manner is obtained by utilizing a GO-SELEX technology, and the largemouth bass virus can be rapidly, sensitively and specifically detected. The aptamer is low in cost, good in stability and easy to modify, can be used as promising substitute molecule of antibody molecule, and is applied to research work in various fields of life science. The invention also provides a microbial molecular biological detection method, namely a method forrapidly and accurately detecting the largemouth bass virus based on an aptamer technology, and a reference is provided for the construction of a subsequent biosensor and the detection of the largemouth bass virus in an actual sample.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an oligonucleotide aptamer for specifically recognizing largemouth bass virus, a screening method and application thereof. Background technique [0002] Largemouth bass (Micropterus salmoides), commonly known as California bass and largemouth bass, belongs to Perciformes, Porcoidei, Cehtrachidae, and Micropterus in fish taxonomy. , is a wide-temperate freshwater famous fish, mainly distributed in the Mississippi River system in California, North America. Because of its advantages such as strong adaptability, large individuals, rapid growth, easy capture, and short breeding cycle, as well as delicious meat, few thorns and no hard bones, and beautiful appearance, largemouth bass has become an important freshwater cultured species in my country. Popular with farmers and consumers. As a global farmed fish, its 2018 production exceeded 435,000 tons, of which China accounted f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/569
CPCC12N15/115C12N15/1048G01N33/56983C12N2310/16C12N2330/31G01N2333/01C12Q2531/107
Inventor 金仁耀杨加成朱勤超冯俊丽潘晓艺
Owner ZHEJIANG GONGSHANG UNIVERSITY
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