Establishment Of Rapid Detection Of Vibrio Fluvialis Based On Nanobody Immunomagnetic Beads Capture-qPCR Technology

Vibrio fluvialis is a Gram-negative facultative anaerobic bacterium that was widely distributed in river water,seawater and harbor water and.It could cause colitis,and its clinical symptoms were very similar to diarrhea caused by V.cholerae.V.fluvialis mainly caused acute gastroenteritis and diarrhea in humans.Many virulence factors could be produced by V.fluvialis,such as diarrhea,nausea,vomiting,low fever,and dizziness.At present,in China,only V.cholerae and V.parahaemolyticus had been highly concerned,while V.fluvialis had not received the attention of relevant departments,but the safety problems caused by V.fluvialis cannot be ignored.Therefore,it was great significance to establish a method for rapid detection of V.fluvialis.At present,the detection methods of V.fluvialis mainly include:selective identification medium,phage diagnostic technology,polymerase chain reaction(PCR),fluorescent antibody technology,loop-mediated isothermal amplification technology,etc.The PCR method was based on specific primers.The reaction was composed of high temperature denaturation,a low temperature annealing and a suitable temperature extension to form a cycle,so that the target gene could be rapidly expanded.The PCR method had the characteristics of high sensitivity and simple operation.Real-time PCR was based on the improvement of PCR.In the PCR reaction,the fluorescent group was devastated,and the PCR product could be quantitatively analyzed.However,the premise of PCR method was the preparation of template.Extracting DNA template from complex sample matrix was??one of the problems faced by researchers.Therefore,this study intended to use immunomagnetic beads to separate V.fluvialis from the sample matrix and establish Immunomagnetic beads combined with real-time PCR to detect V.fluvialis.The main contents of this study are as follows:1.Expression and purification of anti-V.fluvialis nanobodiesThe nanobody fragment was successfully inserted into the expression vector pET25b-CTB by genetic engineering.The prokaryotic expression system was used to obtain the target protein with higher purity,and the activity and specificity of the Nanobody were analyzed by indirect ELISA.Nb70,Nb71,Nb72,Nb73,Nb74 and Nb75 can bind to V.fluvialis,and the OD values of Nb71 and V.fluvialis are significantly higher than those of the other five strains,and they have no binding with other bacterial strains.Well,the stability was verified by heating.The results showed that the antibody remained at 60%activity after heating at 65°C for 60 min.Nb71 has good stability.Therefore,Nb71 was selected for subsequent experiments.2.Establishment of PEI-GA immunomagnetic beads combined with qPCR for rapid detection of V.fluvialisIn this study,a method based on immunomagnetic beads enrichment combined with qPCR for rapid detection of V.fluvialis was established.Eluted V.fluvialis from immunomagnetic beads by heating elution method.And then,SDS lysis method was used to extract V.fluvialis DNA as qPCR template.The standard curve was drawn between CT value and different concentration of V.fluvialis Ma2598.The CT value showed a good linear relationship with the different concentrations of V.fluvialis Ma2598.The linear equation was y=-3.6777x+39.702.R~2=0.9901,the minimum detection limit was 48 cfu/mL.After artificial addition of V.fluvialis Ma2598 to fish samples and water samples.The minimum detection limit in fish samples was 4.8×10~2cfu/mL,and the minimum detection limit in water samples was 48 cfu/mL.The results showed that the method based on PEI immunomagnetic beads combined with qPCR for detection of V.fluvialis was feasible in practical samples.