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Oligonucleotide sequence for detecting vibrio alginolyticus and application thereof

A technology of oligonucleotides and Vibrio alginolyticus, which is applied in the direction of resistance to vector-borne diseases, measurement/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of long cycle, heavy workload, cumbersome operation, etc. Achieve the effects of short preparation cycle, rapid detection and simple operation

Inactive Publication Date: 2011-09-28
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although chemicals and antibiotics can control the 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 to pollute 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, Vibrio detection methods are mainly identified by detecting the physiological and biochemical characteristics of microorganisms according to Bergey's manual. Since there are many items to be tested, this method has a large workload and cumbersome operations; 16SRNA Although molecular biology methods have high accuracy, they need to extract 16SRNA of pathogenic bacteria, the procedures are cumbersome, and there are certain requirements for detection equipment, so it is difficult to achieve rapid detection on the spot; although antiserum prepared by immunological methods can achieve rapid However, since the prepared antiserum is a polyclonal antibody, false positives or false negatives may occur in practical applications. Even if monoclonal antibodies are used, their application is limited due to the high technical requirements for preparation and the long cycle.

Method used

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  • Oligonucleotide sequence for detecting vibrio alginolyticus and application thereof
  • Oligonucleotide sequence for detecting vibrio alginolyticus and application thereof
  • Oligonucleotide sequence for detecting vibrio alginolyticus and application thereof

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Embodiment 1

[0022] The preparation steps of a class of oligonucleotide sequences that can be used for identification and detection of Vibrio alginolyticus in this embodiment mainly include:

[0023] 1. Synthetic oligonucleotide library and primers (synthesized by Shanghai Sangon Biotechnology Co., Ltd.): Oligonucleotide library (SEQ ID No.6): 5′- TCA GTC GCT TCG CCG TCT CCT TC --- -N35----GCA CAA GAG GGA GAC CCC AGA GGG -3′ (N35 is 35 random oligonucleotide sequences)

[0024] Primer 1 (P1): 5′- TCA GTC GCT TCG CCG TCT CCT TC -3′ (SEQ ID No. 7)

[0025] Primer 2 (P2) 5'- CCC TCT GGG GTC TCC CTC TTG TGC-3' (SEQ ID No.8)

[0026] 2. SELEX screening

[0027] The oligonucleotide screening library synthesized above was first combined with the target Vibrio alginolyticus (purchased from the Institute of Microbiology, Chinese Academy of Sciences), and the Vibrio was separated by centrifugation, and then the ssDNA with affinity was isolated from the Vibrio by heating, and then passed asymmetric...

Embodiment 2

[0066] The oligonucleotide sequence has the purpose of identifying and detecting Vibrio alginolyticus: the specific steps are as follows: 1) Take the wound tissue mucus of diseased fish, dissolve it in distilled water and inoculate it into Tryptone Soy Broth (TSB) medium, 30 Cultivate on a shaker at 100 rpm for about 8-10 h at ℃. Then take the bacterial solution and centrifuge, discard the supernatant culture solution, and then dilute to 1×10 with distilled water. 8 / mL to 9×10 8 per mL, and stored at -20°C for later use. Then use the aptamer 8 (SEQ ID No.1) labeled with digoxin at the 5' end to detect according to the previous affinity determination method, and simultaneously make a positive control (using Vibrio alginolyticus as a positive control) and a negative control ( Use sterile distilled water as a negative control), the results showed that the color of the positive control group and the experimental group turned yellow, while the color of the negative control group...

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Abstract

The invention discloses an oligonucleotide sequence for detecting vibrio alginolyticus and application thereof. The oligonucleotide sequence for detecting vibrio alginolyticus comprises four oligonucleotide sequences, such as SEQIDNo.1, SEQIDNo.2, SEQIDNo.3 and SEQIDNo.4, and the vibrio alginolyticus can be identified and detected by one oligonucleotide sequence. The oligonucleotide sequence disclosed by the invention has the characteristics that the detection is rapid, the operation is simple and the stability of an adaptor is higher than that of an antibody, is easy to prepare and has shorter preparation period.

Description

technical field [0001] The invention relates to a class of oligonucleotide sequences that can be used for the identification and detection of Vibrio alginolyticus and applications thereof. Background technique [0002] In the aquaculture industry, about 10% of aquaculture animals die of infectious diseases every year, among which the diseases caused by Vibrio infection account for a considerable proportion. Although chemicals and antibiotics can control the 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 to pollute 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, Vibrio detection methods are mainly identified by detecting the physiological and biochemical characteristics of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCY02A50/30
Inventor 郑江郝聚敏
Owner JIMEI UNIV
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