Aptamer-based quantitative vibrio alginolyticus detection method

Abstract

The invention discloses an aptamer-based quantitative vibrio alginolyticus detection method. The method includes steps: synthesizing aptamers and marking fluorescein; respectively mixing to-be-detected vibrio alginolyticus and vibrio alginolyticus in known concentration with the aptamers; after well mixing, combining vibrio alginolyticus with a detection reagent; after combination is finished, centrifuging, removing supernatant, and washing bacterial precipitates with washing buffer solution; repeating the steps; resuspending the bacterial precipitates with washing buffer solution, and transferring the buffer solution to a new tube; adopting a fluorescence method for determining a fluorescence value of solution in the new tube; performing linear fitting according to the fluorescence valueto obtain a corresponding standard curve and fitting equation, and calculating concentration of the to-be-detected vibrio alginolyticus according to the standard curve. The quantitative detection method for vibrio alginolyticus and inactivated bacteria thereof has advantages of simplicity, convenience, quickness, high recognizability, high repeatability, high accuracy and the like.

Description

technical field [0001] The invention relates to a quantitative detection method, in particular to a method for quantitative detection of Vibrio alginolyticus based on nucleic acid aptamers. Background technique [0002] With the continuous expansion of aquaculture scale, aquaculture animals are infected by various pathogenic microorganisms and cause more and more diseases. One of the important pathogenic bacteria is Vibrio alginolyticus. Although a series of detection methods have been developed for Vibrio alginolyticus and have achieved good results, most of the detection methods cannot be carried out on a large scale due to cumbersome operations, long detection cycles, high costs, and the need for some large precision instruments. Therefore, it is necessary to carry out research on the quantitative detection of Vibrio alginolyticus in the water environment to effectively monitor the number of Vibrio alginolyticus in the water environment and prevent possible outbreaks dis...

AI Technical Summary

Problems solved by technology

Bacterial culture method has a large workload, tedious steps, time-consuming, and large error in the results. It is impossible to distinguish pathogenic bacteria with the same or similar physiological and biochemical characteristics, especially pathogenic bacteria belonging to the same species, which is not conducive to the rapid quantitative detection of pathogenic bacteria.

Antigen-antibody-based quantitative detection technology needs to immunize animals with pathogenic bacteria as antigens to obtain corresponding antibodies or serum. The whole immunization process is not only time-consuming and cumbersome, but also has the following shortcomings: ①Strict preparation requirements and high cost; ②Can be used as antigens The range of animals used for immunization is limited, and the antigens with weak antigenicity or antigens with too close kinship and immunized animals are not easy to produce antibodies; Not strong enough; ④Antibody protein is produced by living cells, which is greatly affected by immunized animals, and the preparation process is difficult to control. When immunizing animals, a large amount of non-specific antibodies are easily produced; The impact of denaturation and loss of activity, storage and transportation conditions are relatively harsh

[0005] The above research reports all show that nucleic acid aptamers have good application prospects in the quantitative detection of Vibrio alginolyticus, but there are few domestic and foreign reports on the quantitative detection of Vibrio alginolyticus using nucleic acid aptamers as recognition molecules

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