Development Of Rapid Detection Technology For Important Pathogenic Bacteria In Mariculture

With the development of the global aquaculture industry,the scale of aquaculture continues to expand,followed by aquatic diseases,resulting in huge economic losses of farmers worldwide,hindered the healthy development of aquaculture industry.Among them,bacterial diseases are one of the most serious diseases in aquaculture industry.According to epidemiological studies in recent years,Vibrio harveyi,Vibrio parahaemolyticus,Vibrio scophthalmi,Vibrio anguillarum and Photobacterium damselae subsp.damselae are common pathogens in mariculture,with strong pathogenicity and widespread epidemic characteristics.In order to diagnose and detect pathogens as soon as possible and reduce the risk of economic loss,rapid detection of multiple pathogens in aquaculture field is one of the important technical requirements for the healthy development of aquaculture industry.The purpose of this study:1.Multiple microfluidic fluorescence quantitative detection method for five bacteria:V.harveyi,V.parahaemolyticus,V.scophthalmi,V.anguillarum and P.damselae subsp.damselae.2.The pir A and pir B genes of V.parahaemolyticus,the pathogen of hepatopancreas necrosis in shrimp,were detected by dual microfluidic quantitative PCR.The one-step rapid detection of important mariculture pathogens by microfluidic fluorescence quantitative PCR was realized,which laid a foundation for the development of multiple microfluidic detection technology of pathogens applied in aquaculture field.1.Establishment of microfluidic fluorescence quantitative PCR method for detection of V.harveyi,V.parahaemolyticus and V.scophthalmi.In this study,specific primers were designed for vhh A gene of V.harveyi,tox R gene of V.parahaemolyticus,lux R gene of V.scophthalmi based on UF-150 microfluidic fluorescence quantitative PCR instrument.By optimizing reaction time and system,microfluidic fluorescence quantitative PCR technology was established for detection of the three kinds of bacteria.The test results are displayed directly on the portable computer.The experimental results showed that the three methods have good specificity.A good linear correlation was found in the range of 10⁹～10⁴copies/μL by drawing standard curves.The sensitivity of the established method to V.harveyi,V.parahaemolyticus and V.scophthalmus were 2.42×10 copies/μL,3.83×10² copies/μL and 2.87×10copies/μL,respectively.Moreover,the detection time is only 26min,which significantly reduces the amplification time of fluorescence quantitative PCR,and has the ability of on-site rapid detection,which lays a foundation for the further development of microfluidic integrated chip for one-step rapid detection of multiple pathogens.2.Establishment of a multiplex microfluidic fluorescent quantitative PCR method for the rapid detection of Mermaid subspecies V.harveyi,V.parahaemolyticus,V.scophthalmi,V.anguillarum and P.damselae subsp.damselae.This research in the laboratory on the basis of existing achievements for the vickers vhh A gene of V.harveyi,tox R gene of V.parahaemolyticus,lux R gene of V.scophthalmi,emp A gene of V.anguillarum and Mcp gene of P.damselae subsp.damselae as a target gene specific primers,by optimizing reaction system and conditions,in microfluidic chip integration,A multiplex microfluidic quantitative PCR technique was developed for simultaneous detection of these five pathogens.The results showed that the optimal reaction system was as follows:2×Taq Pro Universal SYBR q PCR Master Mix 5μL,Primer F/R 1μL,DNA template 2μL,dd H₂O 1μL.The reaction conditions were as follows:95℃for 30 s,95℃for 5 s,61℃for 30 s,and 30 cycles of amplification.The method showed high specificity for V.harveyi,V.parahaemolyticus,V.scophthalmi,V.anguillarum and P.damselae subsp.damsela,and the lowest detection limits for the 5 pathogens were:4.0×10 CFU/m L,2.0×10 CFU/m L,2.0×10² CFU/m L,5.0×10² CFU/m L and 2.0×10 CFU/m L showed high sensitivity,and the average detection time of samples was reduced to about 26 min.The results of this study lay an important foundation for the development of rapid and accurate field detection technology of multiple pathogens in aquaculture.3.Establishment of dual microfluidic fluorescence quantitative PCR for rapid detection of Vp _AHPND.In this study,a strain of V.parahaemolyticus isolated in laboratory that can cause hepatopancreas necrosis of penaeus shrimp was identified by national standard nested PCR and found to carry pir A and pir B genes.Therefore,specific primers were designed for the unique conservative sequences of pir A and pir B genes of Vp _AHPND.By optimizing reaction conditions and reaction system,a double microfluidic fluorescence quantitative PCR rapid detection method was established.The results showed that the established method has good specificity and can accurately distinguish Vp _AHPND from non-Vp _AHPND.The minimum detection limit of this method was 10copies/μL.To provide technical support for rapid diagnosis of hepatopancreas necrosis of prawn.4.Clinical testing and application.Clinical samples were simulated in the laboratory,and the established method was used for detection.Meanwhile,quantitative fluorescence PCR was used in the laboratory to verify the application effect.The accuracy of the established multi-microfluidic fluorescence quantitative PCR method was 96.2%higher than that of the conventional real-time fluorescence quantitative PCR method.It was found that the gills of fish,muscle and gills of shrimp had the best detection effect.The detection results showed that the minimum detection limit was 10⁴ CFU/g for the tissue parts with 5 kinds of pure bacterial solutions,and 10⁵ CFU/g for the tissue parts with 5 kinds of mixed bacterial solutions.It was found that the sensitivity of the established multi-microfluidic fluorescence quantitative PCR method was one order of magnitude lower than that of the real-time fluorescence quantitative PCR method.The average detection time of the samples was only 26 min,which was significantly shorter than the normal real-time quantitative PCR reaction time of 1 h and 40 min.This method can be used for the preliminary diagnosis of pathogen of disease in aquaculture field.