Methods for rapid bacterial DNA isolation suitable for identification of Aeromonas hydrophila via isothermal amplification in aquaculture

BACKGROUND: Russian aquaculture develops rapidly in terms of commercial rainbow trout production that requires massive juvenile stocks. In the last decade, the industry for the production of juvenile trout from fertilized eggs has been formed and is now expanding. Most fish farmers are striving to carry out this stage under a fully controlled water regime in recirculating aquaculture system. Today, the problem of ongoing monitoring of the bacterial pathogens and control of their numbers in such recirculating aquaculture system does not have a solution for practical fish farms. Given that the use of antibiotics is strictly regulated in food production, a project was launched to develop a bacteriophage specifically targeting highly pathogenic bacterial species.

AIM: Study various samples taken from the recirculating aquaculture system to determine the optimal site and method for bacteria collection and nucleic acid extraction.

METHODS: Samples were collected during 2025 at recirculating aquaculture system for rainbow trout Oncorhynchus mykiss in the Leningrad Region and the Republic of Karelia. The bacteria studied in this article are Aeromonas hydrophila. The bacterial content was assessed using traditional bacterial cultivation methods. DNA was extracted from various sample types and subjected to isothermal amplification targeting A. hydrophila. For samples confirmed to contain A. hydrophila, additional processing methods were employed following initial lysis in a buffer composed SDS and NaOH. Both chemical techniques, such as precipitation with alcohols and nanoparticles, and physical methods, including heating and syringe pipetting, were utilized in this procedure.

RESULTS: The study found that certain chemical methods (nanoparticle precipitation) for isolating bacterial DNA from the pre-lysed samples were just as effective as physical methods (syringe pipetting and heating). The most informative sample types for pathogen detection sites from the recirculating aquaculture system were swabs taken from pipes of the fish tanks drain.

CONCLUSION: The ability to obtain microorganism identification results using nucleic acid amplification methods outside the laboratory makes them highly promising for use as rapid diagnostics for bacterial pathogens in practical aquaculture. Following the completion of sample collection and the creation of a collection of pathogenic organisms, the development plan includes the development of a genetically modified bacteriophage for bacterial population control.