Pseudomonas plecoglossicida aptamers and screening method therefor

A Pseudomonas mutans, screening method technology, applied in biochemical equipment and methods, DNA preparation, DNA / RNA fragments, etc., can solve problems such as inability to fully explain pathogenicity

Pending Publication Date: 2021-08-20
JIMEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, although there are many research reports on visceral white spot disease of large yellow croaker, they mainly focus on pathological and morphological analysis. Although its pathogenic mechanism is related to extracellular products, it cannot fully explain its path

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  • Pseudomonas plecoglossicida aptamers and screening method therefor
  • Pseudomonas plecoglossicida aptamers and screening method therefor
  • Pseudomonas plecoglossicida aptamers and screening method therefor

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Embodiment Construction

[0036] The present invention takes Pseudomonas mutans as an object, adopts reverse screening and other SELEX technologies to screen nucleic acid aptamers that can specifically recognize Pseudomonas mutans, and then verifies the affinity of the nucleic acid aptamers.

[0037] 1 SELEX screening of nucleic acid aptamers

[0038] (1) Bacteria treatment: take the cultured Pseudomonas mutans centrifuge tube, centrifuge at 6000r / min for 5min, discard the supernatant, wash the bacteria 3 times, add sterile culture medium and mix evenly, measure the OD value of the bacteria solution, Then take the bacteria content of about 4 × 10 8 Centrifuge the Pseudomonas mutans solution at 6000r / min for 5min, discard the supernatant, wash the bacterial pellet three times with 0.9% normal saline, wash the bacterial pellet once with 1×binding buffer, and finally add 100μL of 2×binding buffer Resuspended.

[0039] (2) Binding: take ssDNA random library, dilute to 2μmol / L100μL with 2×binding buffer, ...

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Abstract

The invention relates to aptamers and particularly relates to Pseudomonas plecoglossicida aptamers and a screening method therefor. Sequences of the aptamers are represented by SEQ. NO. 1-5, the aptamers capable of specifically identifying and inhibiting the growth of Pseudomonas plecoglossicida are screened by employing SELEX technologies such as inverse screening, and the influence on the Pseudomonas plecoglossicida caused by the aptamers is characterized through measuring affinity. According to the Pseudomonas plecoglossicida aptamers and the screening method therefor, high-frequency sequences can be objectively and effectively classified, and thus, an objective and effective basis is provided for subsequent accurate selection and verification on the aptamers; and the method is simple and is convenient in operation, so that the blindness of verification is lowered greatly, and the screening efficiency is increased. Five aptamers #1, #2, #3, #4 and #5 with highest relative important indexes are picked from a screened result, the affinity of these aptamers to 28-DEG-C Pseudomonas plecoglossicida is higher than that of other bacteria, and thus, these aptamers have relatively good affinity and specificity to the 28-DEG-C Pseudomonas plecoglossicida.

Description

technical field [0001] The invention relates to a nucleic acid aptamer, in particular to a Pseudomonas mutans aptamer and a screening method thereof. Background technique [0002] Pseudomonas is widely distributed in forest soil, river water and organisms, and is one of the common bacterial species in the world. At present, according to the sequence of 16sRNA, Pseudomonas can be divided into two categories. Pseudomonas fluorescens, Pseudomonasputida and Pseudomonasstutzeri, the second type is Pseudomonaspertucinogena. Pseudomonas not only infects humans and seriously affects human health and life, but they are also pathogenic bacteria in farmed economic fish. According to reports, the fish species infected by Pseudomonas putida are mainly concentrated in sweetfish, rainbow trout and The fish species infected by Pseudomonas aeruginosa are mainly concentrated in carp and tilapia. The fish species infected by Pseudomonas plecoglossicida are mainly concentrated in sweetfish, ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10
CPCC12N15/115C12N15/1048C12N2310/16
Inventor 郑江许净鄢庆枇江兴龙
Owner JIMEI UNIV
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