A group of oligonucleotide sequences capable of identifying Vibrio harveyi and Vibrio alginolyticus synchronously and preparation method of the oligonucleotide sequences

A technology of Vibrio harveii and Vibrio alginolyticus, applied in the field of oligonucleotide sequences, can solve the problems of easy generation of drug-resistant strains, false positives, long cycle and the like

Active Publication Date: 2012-07-25
JIMEI UNIV
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  • Claims
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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 16SrRNA has high accuracy, it needs to extract the 16SrRNA of pathogenic bacteria, which requires a high level of professional skills and certain requirements for detection equipment, so it is difficult to achieve rapid detection on the spot; Although serum can achieve rapid detection, because the prepared antiserum is a polyclonal antibody, false positives or false negatives may occur in practical applications. also subject to certain restrictions

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  • A group of oligonucleotide sequences capable of identifying Vibrio harveyi and Vibrio alginolyticus synchronously and preparation method of the oligonucleotide sequences
  • A group of oligonucleotide sequences capable of identifying Vibrio harveyi and Vibrio alginolyticus synchronously and preparation method of the oligonucleotide sequences
  • A group of oligonucleotide sequences capable of identifying Vibrio harveyi and Vibrio alginolyticus synchronously and preparation method of the oligonucleotide sequences

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Experimental program
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Effect test

Embodiment 1

[0083] The preparation steps of a set of oligonucleotide sequences capable of simultaneously identifying Vibrio harveyi and Vibrio alginolyticus in this embodiment mainly include:

[0084] 1. Preparation of Vibrio liquid

[0085] Wash the colony of Vibrio harveyi and Vibrio alginolyticus from the slope with normal saline, centrifuge at 6000rpm, discard the supernatant, resuspend and dilute the bacterial solution with normal saline to the following concentration range: 1×10 8 ~9×10 8 pc / ml, stored at -20°C for later use;

[0086] 2. SELEX screening of aptamers

[0087] 2.1 Combination and isolation of ssDNA with Vibrio harveyi

[0088] Take 4 μL of 100 μM ssDNA oligonucleotide library, dilute to 100 μl with 2× binding buffer, denature at 95°C for 5 minutes, add 100 μl of Vibrio harveii bacteria solution restored to natural room temperature after 10 minutes in ice bath, combine on a shaking table for 30 minutes, and then Centrifuge at 6000rpm for 5min, discard the supernatan...

Embodiment 2

[0131] The oligonucleotide sequence has the purpose of identifying and detecting Vibrio harveyi: the specific steps are as follows: 1) get the wound tissue mucus of the diseased fish, dissolve it in distilled water and inoculate it in the tryptone soybean broth medium (TSB) medium, Incubate on a shaker at 100 rpm at 30°C for 8-10 hours. Then take the bacterial solution and centrifuge, discard the supernatant culture solution, then dilute it with normal saline to 1×108~9×108 cells / mL, and store it at -20°C for later use. Then carry out affinity detection according to the method of the affinity-specific verification of the step 4.3 in the embodiment 1 with the aptamer C7 (SEQ ID No.1) that 5 ' end is marked with digoxin; Alginolyticus and Vibrio alginolyticus were used instead of the test bacteria solution, and the affinity test was carried out in the same way to obtain two positive controls; sterile distilled water was used instead of the test bacteria solution, and the affinit...

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Abstract

The invention discloses a group of oligonucleotide sequences capable of synchronous identification of Vibrio harveyi and Vibrio alginolyticus and a preparation method of the oligonucleotide sequences, and relates to identification and detection of Vibrio harveyi and Vibrio alginolyticus. The oligonucleotide sequences comprise SEQ ID No.1-4. The preparation method comprises the following steps: synthesizing an ssDNA (single-stranded deoxyribonucleic acid) oligonucleotide library (5'-TCA GTC GCT TCG CCG TCT CCT TC----N35----GCA CAA GAG GGA GAC CCC AGA GGG-3') for screening; separately mixing the oligonucleotide library with Vibrio harveyi and Vibrio alginolyticus, and carrying out SELEX (systematic evolution of ligands by exponential enrichment) screening; cloning and sequencing the aptamer-rich library; and selecting high-copy ssDNA in the sequencing result, verifying specificity of affinity, and obtaining the corresponding aptamers.

Description

technical field [0001] The invention relates to oligonucleotide sequences, in particular to a group of oligonucleotide sequences capable of simultaneously identifying Vibrio harveyi and Vibrio alginolyticus and a preparation method thereof. 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 method of Vibrio is mainly based on the Berg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10C12R1/63
Inventor 郑江郝聚敏
Owner JIMEI UNIV
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