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Specific sequence-based detection primers, kits, detection methods and development methods for Aeromonas victoria

A technology of Aeromonas victoria and detection reagents, applied in the field of molecular biology, can solve the problems of patient persistence, death, misdiagnosis, etc.

Inactive Publication Date: 2021-05-18
HAINAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Therefore, generally speaking, the detection method of Aeromonas victorii is still in the lagging stage, so that aquatic animals, especially humans, are often misdiagnosed when they are infected with pathogenic bacteria, often causing serious consequences - a large number of deaths of farmed aquatic animals and patients persistent illness or even death

Method used

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  • Specific sequence-based detection primers, kits, detection methods and development methods for Aeromonas victoria
  • Specific sequence-based detection primers, kits, detection methods and development methods for Aeromonas victoria
  • Specific sequence-based detection primers, kits, detection methods and development methods for Aeromonas victoria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Mining of Aeromonas verkis species-level specific sequences

[0089] Using bioinformatics methods, by writing python program scripts, according to figure 1 As shown in the workflow, all protein sequences included in NCBI are analyzed.

[0090] figure 1 The flow shown includes the following steps:

[0091] (a) Obtain data subset 1 and data subset 2 from the NCBI database, wherein the data subset 1 includes all protein sequences of Aeromonas (genus level) included in the NCBI database, but does not include Aeromonas vickeris Any protein sequence of bacteria (level of species), said data subset 2 includes all protein sequences of Aeromonas victorii (level of species) included in the NCBI database; and in data subset 1 and data subset 2 Blast alignment of the sequence of the sequence to obtain sequence subset A and sequence subset a (this is equivalent to screening species-specific protein sequences at the genus level), wherein the sequence subset A cannot b...

Embodiment 2

[0100] Embodiment 2. test material, test method and equipment

[0101] 1. Test materials and equipment

[0102] Strains:

[0103] Bacillus subtilis, Vibrio parahaemolyticus, Aeromonas veronii, Edwardsiella tarda, Vibrio alginolyticus, Escherichia coli coli), Pseudomonas aeruginosa (Pseudomonasaeruginosa) are preserved by our laboratory.

[0104] Media and reagents:

[0105] Bacterial Genomic DNA Rapid Extraction Kit (Cat. No. N1152) and PCR Reagents were purchased from Guangzhou Dongsheng Biotech Co., Ltd. (Dongsheng Biotech Co., Ltd);

[0106] Tryptone, yeast extract, agar powder, and agarose were purchased from Guangdong Weijia Biological Co., Ltd.;

[0107] 1) LB liquid medium, containing the following substances: tryptone 1.0g, yeast extract 0.5g, NaCl 0.5g, distilled water 100mL, adjust pH to 7.2 with 10mol / L NaOH, autoclave at 121°C for 20min, store at 4°C spare.

[0108] 2) LB solid medium, containing the following substances: tryptone 1.0g, yeast extract 0.5g, ...

Embodiment 3

[0131] Example 3. Design primers and primer screening based on Aeromonas verkis species-level specific sequence

[0132] Using Primer Premier 5.0 and DNAMAN software, multiple sets of primers were designed for the specific nucleic acid sequence obtained in Example 1, and the designed primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0133] Using the genomic DNA of Aeromonas verkisii as a template, and using different primer pairs designed and synthesized, PCR reactions were carried out according to the method in Example 2. PCR amplification products were analyzed by 30g / L agarose gel electrophoresis.

[0134] According to the results of gel electrophoresis (not shown), a single primer pair with clear amplified bands was selected for further analysis of primer specificity. A total of two pairs of primers were obtained, the sequences of which are shown in SEQ ID No.1-4, and the sequence information of the primers is shown in Table 2. The amplified product...

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Abstract

The present invention relates to the pair of primers that are used to detect Aeromonas verkirea, the nucleotide sequence of the forward primer of the pair of primers is selected from SEQ ID NO.1 and / or SEQ ID NO.3, the pair of primers The nucleotide sequence of the reverse primer is selected from SEQ ID NO.2 and / or SEQ ID NO.4. The present invention also relates to a reagent, a kit comprising the primer pair or primer pair combination and its application in detecting Aeromonas verkirea. The present invention also relates to a method of developing primers for the detection of Aeromonas vernerii.

Description

technical field [0001] The present invention relates to a specific sequence-based detection primer for Aeromonas virtii, a reagent comprising the primer, a kit, a detection method for Aeromonas virtii and a method for developing a detection primer for Aeromonas virtii The method belongs to the field of molecular biology. Background technique [0002] Aeromonas veronii (Aeromonas veronii), also known as Aeromonas verona, Aeromonas veronii, Aeromonas vanlon, is a Gram-negative rod-shaped bacterium that is non-spore-forming Facultative anaerobic type. The fungus is scattered and commonly found in fresh water, sewage, soil and even sea water. The two ends are blunt and round, the size is about 0.3-0.7 μm×1.2-2.5 μm, and it has flagella and can move (Kirov SM, Tassell BC, Semmler AB, O'Donovan LA, Rabaan AA et al. "Lateral flagella and swarming motility in Aeromonas species" [J].J Bacteriol.2002,184(2):547-555). [0003] Aeromonas verdeii is a virulent pathogenic bacteria. The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/6811C12Q1/04C12N15/11
CPCC12Q1/6811C12Q1/6851C12Q1/689C12Q2561/101C12Q2531/113C12Q2537/165
Inventor 刘柱马香唐燕琼唐鸿倩胡新文杜明伦
Owner HAINAN UNIV