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Quick detection method for scallop pathogenic vibrio splendidus

A technology of Vibrio brilliant and detection method, which is applied in the field of aquatic organisms, can solve problems such as being susceptible to interference, unstable results, and low sensitivity of enzyme-linked immunosorbent assay, achieving the effect of ensuring accuracy, reliability and wide detection range

Active Publication Date: 2013-12-25
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

However, these methods have many shortcomings and deficiencies: physiological and biochemical detection is time-consuming and labor-intensive, and the results are unstable; fluorescent antibody technology requires more expensive equipment; enzyme-linked immunosorbent assay is not sensitive and is susceptible to interference; rRNA gene It is very conservative in species, making the technical specificity not strong; it is difficult for ordinary PCR methods to meet the requirements of quantitative analysis

Method used

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  • Quick detection method for scallop pathogenic vibrio splendidus
  • Quick detection method for scallop pathogenic vibrio splendidus
  • Quick detection method for scallop pathogenic vibrio splendidus

Examples

Experimental program
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Effect test

experiment example 1

[0027] Experimental Example 1: Specificity Analysis of Primers for Quantitative Detection of Vibrio splendidus:

[0028] The metalloprotease gene in Vibrio candidiasis can be specifically detected by using the primers for quantitative detection of Vibrio candidiasis.

[0029] Vibrio resplendent, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa and Pseudomonas putida were cultured to extract whole genome DNA as PCR reaction template. The general PCR reaction was carried out by using the primers for the quantitative detection of metalloprotease of Vibrio resplendent and the above-mentioned template. The reaction conditions were pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, renaturation at 60°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; extension at 72°C for 10 minutes. After the PCR reaction, the products were detected on agarose gel. The results showed that there w...

experiment example 2

[0030] Experimental Example 2: Sensitivity Analysis of Primers for Quantitative Detection of Vibrio splendidus:

[0031] Vibrio cantilever can be detected with high sensitivity by using the primer for quantitative detection of Vibrio candidiasis metalloproteinase.

[0032] Culture Vibrio splendidus with 2216E liquid culture based on shaking at 18°C ​​for 12 hours, and use the method of plate counting to determine the concentration of the bacterial liquid to be 4×10 9 CFU / ml, use sterile normal saline to dilute the bacterial solution of Vibrio splendidus in a multiple of 10, and the concentrations are 4×10 8 CFU / ml, 4×10 7 CFU / ml, 4×10 6 CFU / ml, 4×10 5 CFU / ml, 4×10 4 CFU / ml, 4×10 3 CFU / ml, 4×10 2 CFU / ml, 4×10 1 CFU / ml, 4CFU / ml. Use the above dilutions of bacteria as templates for common PCR reactions. The reaction conditions are 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 60°C refolding for 30 seconds, 72°C extension for 30 seconds, 30 cycles;...

Embodiment 3

[0034] Example 3: Detection of the proportion of Vibrio splendidus in the culture environment of Dalian scallops

[0035] Water samples were collected from the Dalian scallop culture environment in March, May, July, August, September, and November respectively. The water samples passed through a 0.22 μm filter membrane to enrich the microorganisms in the environment, and the total DNA on the filter membrane was extracted. , to determine the concentration of DNA in environmental samples in each month.

[0036] Before the fluorescent quantitative PCR reaction, the DNA concentration of the environmental samples in each month was diluted to 1ng / μl, and the diluted samples were used for fluorescent quantitative PCR with 16SrDNA and metalloprotease gene primers respectively, and the reaction conditions were pre-denaturation at 95°C for 30 seconds; Denaturation at 95°C for 5 seconds, annealing / extension at 60°C for 30 seconds, collection of SYBR Green fluorescence, 40 cycles. Accord...

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Abstract

The invention belongs to the technical field of aquatic organisms and in particular relates to a quick detection method for scallop pathogenic vibrio splendidus. Total DNA (deoxyribonucleic acid) of bacterial genomes in a filtered seawater sample to be detected is taken as a template, fluorescent quantitative PCR (polymerase chain reaction) amplification is carried out by a bacterial 16SrDNA conserved region and a specific primer of a pathogenic vibrio splendidus metalloproteinases gene, and the scallop pathogenic vibrio splendidus in the culture environment is quantitatively analyzed and detected; the detection method comprises the steps of extracting the DNA of the bacterial genomes and carrying out fluorescent quantitative PCR detection. The method has the advantages that the quickness is achieved, the specificity is good, the bacterial density detection range is wide, and a quantitative result can be close to a real situation.

Description

technical field [0001] The invention belongs to the technical field of aquatic organisms, in particular to a rapid detection method for scallop pathogenic Vibrio splendidus. Background technique [0002] Vibrio splendidus (Vibrio splendidus) widely exists in the marine environment and is the main pathogenic bacteria causing infection and disease of aquatic animals. It can cause tissue ulceration, abnormal development and mass death of seedlings of aquatic shellfish, which is very harmful. Existing studies have shown that extracellular metalloprotease (Metalloprotease) is the main pathogenic factor of Vibrio splendidus endangering aquatic animals, that is, the source of infection and pathogenicity. The metalloprotease gene is located on chromosome 1 of the Vibrio candidiasis genome and is a single-copy gene, which is convenient for quantitative analysis. [0003] Currently, detection methods for Vibrio resplendent mainly include physiological and biochemical detection method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 宋林生邱丽梅刘瑞张峘
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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