Six oligonucleotide sequences and application thereof

An oligonucleotide and sequence technology, applied in the field of identification and detection of Vibrio alginolyticus, can solve the problems of long cycle, false positive, cumbersome operation, etc.

Inactive Publication Date: 2013-04-24
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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  • Six oligonucleotide sequences and application thereof
  • Six oligonucleotide sequences and application thereof
  • Six oligonucleotide sequences and application thereof

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Experimental program
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Effect test

Embodiment 1

[0095] The preparation steps of the 6 oligonucleotide sequences that can be used for the identification and detection of inactivated Vibrio alginolyticus in this embodiment mainly include:

[0096] 1. Synthesis of ssDNA oligonucleotide library for screening (5′-TCA GTC GCT TCG CCG TCT CCT TC----N35----GCACAAGAG GGAGAC CCCAGAGGG-3′): provided by Shanghai Sangon Biotechnology Co., Ltd. Company chemical synthesis.

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

[0098] 2.1 Preparation of inactivated Vibrio alginolyticus bacterial solution

[0099] Wash Vibrio alginolyticus colonies cultivated 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 in a water bath at 62.5°C for 1 hour, wash with normal saline three times after centrifugation, Suspend the extinguished bacteria pellet with 2×binding buffer and store at 4°C.

[0100] 2.2 Combination with Vibrio ...

Embodiment 2

[0145] Any one of the six oligonucleotide sequences has the purpose of identifying and detecting inactivated Vibrio alginolyticus: the specific steps are as follows:

[0146] 1) Take the aquaculture water body infected with vibriosis, dissolve it in distilled water, inoculate it into tryptone soybean broth (TSB) medium, and cultivate it on a shaker at 100 rpm at 30°C for about 8-10 hours. Then take the bacterial liquid and centrifuge it, discard the supernatant culture liquid, then add physiological saline containing 6% formaldehyde in a water bath at 62.5°C for 1 hour, wash with physiological saline for 3 times after centrifugation, and then suspend the inactivated bacterial precipitate with 2× binding buffer to obtain The inactivated bacterial solution to be tested should be stored at 4°C. Take the inactivated bacterial solution to be tested, and use the aptamer #7 (SEQ ID No.1) labeled with digoxin at the 5′ end to detect the tested inactivated bacterial solution according ...

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Abstract

The invention relates to six oligonucleotide sequences and an application thereof, relating to the recognition detection of vibrio alginolyticus. The invention provides six oligonucleotide sequences capable of inactivating the recognition detection of vibrio alginolyticus, a preparation method and an application thereof. The oligonucleotide sequence has the characteristics of being quick in detection, simple in operation, superior to that of antibody and the like in stability, and the preparation method is easy and relatively short in preparation period. The six oligonucleotide sequences comprise oligonucleotide sequences of SEQ ID No.1-6, and the recognition detection of vibrio alginolyticus can be inactivated by adopting one of the oligonucleotide sequences. The preparation method comprises the following steps: synthesizing an ssDNA oligonucleotide library used for filtering; carrying out SELEX filtering after the oligonucleotide library is mixed with inactivated vibrio alginolyticus till the affinity is not raised obviously; then cloning and sequencing; and selecting a highly copied ssDNA thereof to carry out verification of affinity specificity and determination of affinity constants so as to obtain corresponding an aptamer. The six oligonucleotide sequences can be used for recognition detection of inactivated vibrio alginolyticus.

Description

technical field [0001] The invention relates to the identification and detection of vibrio alginolyticus, in particular to six oligonucleotide sequences which can be used for the identification and detection of vibrio alginolyticus and the application 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 main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 郑江汤学敏李玉宝
Owner JIMEI UNIV
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