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Oligonucleotide sequence as well as preparation method and application thereof

An oligonucleotide and oligonucleotide library technology, applied in biochemical equipment and methods, DNA preparation, microorganism-based methods, etc., can solve the problems of application limitations, false positives, and high technical requirements for preparation

Active Publication Date: 2015-03-04
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although chemicals and antibiotics can control its infection, drug prevention and control often have adverse consequences, such as accumulation and residue in aquatic animals, easy to produce drug-resistant strains and easy pollution of the environment. Therefore, a rapid detection technology for pathogenic Vibrio is established to Early prevention and control of the occurrence and prevalence of diseases is still the main means at present
[0003] At present, the detection method of Vibrio is mainly based on the Bergey's Bacteria Identification Manual, through the detection of the physiological and biochemical characteristics of microorganisms. Since there are many items to be tested, this method has a large workload and cumbersome operation.
Although the molecular biology method of 16SRNA has high accuracy, it needs to extract 16SRNA of pathogenic bacteria. Realize rapid detection, but because the prepared antisera are polyclonal antibodies, false positives or false negatives may occur in practical applications, even if monoclonal antibodies are used
Due to the high requirements of the preparation technology and the long cycle, its application is limited

Method used

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  • Oligonucleotide sequence as well as preparation method and application thereof
  • Oligonucleotide sequence as well as preparation method and application thereof
  • Oligonucleotide sequence as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0095] The preparation steps of the oligonucleotide sequence that can be used for the identification and detection of inactivated Vibrio harveyi in this embodiment mainly include:

[0096] 1. Synthesis of ssDNA oligonucleotide library for screening (5′-TCA GTC GCT TCG CCG TCT CCT TC----N35----GCA CAA GAG GGA GAC CCC AGA GGG-3′): by Shanghai Sheng Synthesized by Gongbio Engineering Technology Co., Ltd.

[0097] 2. SELEX screening: the specific steps are as follows:

[0098] 2.1 Preparation of inactivated Vibrio harveyi liquid

[0099] Wash Vibrio harveyi colonies cultured for 24 hours from the slope with normal saline, centrifuge at 6000rpm for 5 minutes, discard the supernatant, then add normal saline containing 6% formaldehyde and place in a water bath at 62.5°C for 1 hour, wash with normal saline three times after centrifugation , then suspend the inactivated bacterial pellet with 2×binding buffer, and store at 4°C.

[0100] 2.2 Combination with Vibrio

[0101] Take 4 μL...

Embodiment 2

[0143] This oligonucleotide sequence has the purpose of identifying and detecting inactivated Vibrio harveyi: the specific steps are as follows:

[0144] 1) Take the aquaculture water body infected with vibriosis, dissolve it in distilled water, inoculate it into a tryptone soybean broth (TSB) medium, and culture it on a shaker at 100 rpm at 30° C. for about 8 to 10 hours. Then take the bacterial liquid and centrifuge it, discard the supernatant culture liquid, then add physiological saline containing 6% formaldehyde and place it in a water bath at 62.5°C for 1 hour. The inactivated bacterial solution to be tested should be stored at 4°C. Take the inactivated bacterial liquid to be tested, and use the aptamer I9 (SEQ ID No.1) labeled with digoxin at the 5' end to detect the inactivated bacterial liquid to be tested according to the affinity determination method in step 3.3; Vibrio vibrio was used instead of the test bacteria solution, and it was also tested according to the a...

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Abstract

The invention provides an oligonucleotide sequence as well as a preparation method and application thereof, and relates to the identification and detection of inactivated Vibrio harveyi. The oligonucleotide sequence has the characteristics of quickness in detection, high simplicity in operation, higher stability than the stability of antibodies, simple preparation method, short preparation period, and so on. The oligonucleotide sequence is SEQ ID No. 1 which can be used to identify and detect the inactivated Vibrio harveyi. The preparation method comprises the following steps: synthesizing an ssDNA oligonucleotide library for screening; mixing the ssDNA oligonucleotide library with the inactivated Vibrio harveyi and screening the mixture by using the SELEX technology until the affinity no longer increases obviously; carrying out clone sequencing and selecting high copy ssDNA to undergo affinity and specificity verification and affinity constant determination, thereby obtaining the corresponding aptamer; The oligonucleotide sequence can be used to identify and detect the inactivated Vibrio harveyi in various samples.

Description

technical field [0001] The invention relates to the identification and detection of Vibrio harveyi, in particular to an oligonucleotide sequence which can be used for the identification and detection of inactivated Vibrio harveyi as well as its preparation method and application. Background technique [0002] In the aquaculture industry, about 10% of aquaculture animals die from infectious diseases every year, among which the diseases caused by Vibrio infection account for a considerable proportion. Although chemicals and antibiotics can control its infection, drug prevention and control often have adverse consequences, such as accumulation and residue in aquatic animals, easy to produce drug-resistant strains and easy pollution of the environment. Therefore, a rapid detection technology for pathogenic Vibrio is established to Early prevention and control of the occurrence and prevalence of diseases is still the main means at present. [0003] At present, the detection meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68C12Q1/04C12R1/01
Inventor 郑江鄢庆枇郝聚敏唐花
Owner JIMEI UNIV
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