Establishment And Application Of Rapid Detection Techniques For Two Marine Fish Pathogens

Bacterial diseases is one of the most important diseases in marine culture fish.However,the traditional microbiological methods needed to identify the pathogen are often limited by the site and time.In recent years,molecular biology technology has developed rapidly,and the derived molecular diagnosis scheme has stronger specificity and sensitivity,which is also an effective method for rapid detection of the pathogen at present.In this study,Vibrio rotiferianus and Photobacterium damselae subsp.damselae were selected as the objects,and the microfluidic quantitative PCR and RPA detection methods were successively established.The main results are as follows:1.Establishment of microfluidic quantitative PCR for detection of Vibrio rotiferianus based on tox R geneThe highly conserved region of Vibrio rotiferianus tox R gene was selected as the target fragment for the specific primers design.The recombinant plasmid was constructed based on the primers for establish a fluorescence quantitative PCR detection method for V.rotiferianus.And UF-150 Genechecker microfluidic quantitative real-time PCR instrument was used as a platform to develop a microfluidic quantitative real-time PCR detection method for V.rotiferianus with the optimize reaction system and condition.The results indicated that,this method can specifically amplify tox R gene target fragment,and the minimum detection for V.rotiferianus was 1.34×10~0 copies/μL.Artificial infection detection results show that the minimum detected constribution for V.rotiferianus in fish tissues was 1.34×10~3 cfu/g.The results can be visually interpreted and the detection time was less than 42 min.This method has prominent advantages of strong specificity,high sensitivity and low site requirements.2.Establishment of microfluidic fluorescence quantitative detection technology for pathogenic strains of Photobacterium damselae subsp.damselae based on Mcp gene and Dly geneThe highly conserved region of Photobacterium damselae subsp.damselae Mcp gene was selected as the target fragment for the specific primers design.The recombinant plasmid was constructed based on the primers to establish a fluorescence quantitative PCR detection method for PDD.At the same time,based on the virulence gene Dly of Photobacterium damselae subsp.damselae were successfully distinguished strong and weak pathogenic strains.And UF-150 Genechecker microfluidic quantitative real-time PCR instrument was used as a platform to develop a microfluidic quantitative real-time PCR detection method for PDD with the optimize reaction system and condition.The results indicated that,this method can specifically amplify Mcp gene and Dly gene target fragment,and the minimum detection for PDD was 1.0×10~1 copies/μL.Artificial infection detection results show that the minimum detected constribution for PDD in fish tissues was 1.0×10~3 cfu/g.The results can be visually interpreted and the detection time was less than 23 min.This method has prominent advantages of strong specificity,high sensitivity and low site requirements.3.Development of rapid detection of Vibrio rotiferianus and Photobacterium damselae subsp.damselae based on RPAThe single copy tox R gene of V.rotiferianus and the single copy tox R gene of P.damselae subsp damselae were used as the detection target.The RPA detection technology based on tox R gene and Mcp gene based on MCP gene were established respectively by the RPA detection technology.The sensitivity,specificity and stability of the methods were analyzed,and the possibility of its application in field detection was evaluated.The results showed that the sensitivity of RPA detection methods was higher,but it was slightly lower than that of microfluidic quantitative PCR.The RPA primers of tox R gene of V.rotiferianus and Mcp gene of P.damselae subsp damselae were highly specific,and non-specific reaction with non-objective strains such as Vibrio harveyi and Vibrio parahaemolyticus was found.This study proved that this method could replace RT-PCR technology and be used in the research and development of field detection technology.