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Three oligonucleotide sequences for identification and detection of vibrio alginolyticus as well as preparation method and application thereof

A technology of Vibrio alginolyticus and oligonucleotides, applied in biochemical equipment and methods, DNA preparation, and resistance to vector-borne diseases, etc., can solve problems such as long cycle time, false positives, and cumbersome operations

Active Publication Date: 2012-01-25
JIMEI UNIV
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  • 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 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 high preparation technology requirements and long cycle

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  • Three oligonucleotide sequences for identification and detection of vibrio alginolyticus as well as preparation method and application thereof
  • Three oligonucleotide sequences for identification and detection of vibrio alginolyticus as well as preparation method and application thereof
  • Three oligonucleotide sequences for identification and detection of vibrio alginolyticus as well as preparation method and application thereof

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Embodiment 1

[0090] The preparation steps of the three oligonucleotide sequences that can be used for the identification and detection of Vibrio alginolyticus in this embodiment mainly include:

[0091] 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.

[0092] 2. SELEX screening: the corresponding screening process is as follows figure 1 As shown, the specific steps are as follows:

[0093] 2.1 Preparation of Vibrio alginolyticus liquid

[0094] Wash the Vibrio alginolyticus colonies cultivated for 24 hours from the slope with normal saline, centrifuge at 6000rpm for 5 minutes, discard the supernatant, resuspend and dilute the bacterial solution with normal saline to the following concentration range: 1×10 8 / mL to 9×10 8 1 / ml, then take the bacterial solution and divide it into 20 tubes, each with 1ml of the bacterial so...

Embodiment 2

[0142] The oligonucleotide sequence has the purpose of identifying and detecting Vibrio alginolyticus: the specific steps of use are as follows: 1) get the wound tissue mucus of diseased fish, dissolve it in distilled water and inoculate it in the tryptone soybean broth medium (TSB) medium, 30 Cultivate on a shaker at 100 rpm for about 8-10 hours at ℃. Then take the bacterial solution and centrifuge, discard the supernatant culture solution, and then dilute to 1×10 with normal saline. 8 ~9×10 8 per mL, stored at -20°C for later use. Then use the aptamer 46 complementary (SEQ ID No.1) labeled with digoxin at the 5' end to detect according to the affinity determination method in the preparation step 5.3, and simultaneously make a positive control (using Vibrio alginolyticus as a positive control) and Negative control (use aseptic distilled water as negative control), the result shows positive control group and experimental group color turn yellow, and negative control group co...

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Abstract

The invention discloses three oligonucleotide sequences for identification and detection of vibrio alginolyticus as well as a preparation method and an application thereof and relates to identification and detection of vibrio alginolyticus. The oligonucleotide sequences comprise SEQ ID No. 1-3 (sequence identity number 1-3). The 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; mixing the oligonucleotide library with the vibrio alginolyticus and then performing SELEX (systematic evolution of ligands by exponential enrichment) screening; performing cloning and sequencing on an aptamer-enhanced library after completing the SELEX screening; performing interception in different lengths on high-copy ssDNA emerged in the sequencing result so as to get a series of new sequences, and further constructing complementary sequences of the new sequences; and performing affinity and specificity verification on the obtained new sequences and the complementary sequences thereof so as to get corresponding aptamers.

Description

technical field [0001] The invention relates to the identification and detection of Vibrio alginolyticus, in particular to three oligonucleotide sequences that can be used for the identification and detection of Vibrio alginolyticus and 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 method o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/115C12N15/10
CPCY02A50/30
Inventor 郑江郝聚敏苏永全
Owner JIMEI UNIV
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