Establishment Of Visual And Rapid Detection Methods For Nocardia Seriolae And Streptococcus Agalactiae

Nocardia seriolae and Streptococcus agalactiae are important bacterial pathogens of fish,seriously endangering the healthy and green development of aquaculture.The market urgently needs methods that can quickly detect these two fish pathogens on the spot.Therefore,based on recombinase polymerase amplification(RPA)technology,we have established a lateral flow dipstick type RPA(RPA-LFD)and real-time fluorescence RPA visual rapid detection method for N.seriolae and S.agalactiae,laying an important product foundation for the development of on-site rapid diagnostic reagents for N.seriolae and S.agalactiae.The main results achieved are as follows:1.Establishment of a visual and rapid detection method for N.seriolae by RPA-LFD and real-time fluorescence RPABased on the conservative sequence of the 16S-23S r RNA internal transcribed spacer(16S-23S r RNA ITS)of N.seriolae,we have established a rapid visual detection method for N.seriolae using RPA-LFD and real-time fluorescence RPA.Subsequently,we screened the optimal reaction conditions,verified the specificity,and evaluated the sensitivity of the established detection method.RPA-LFD can achieve rapid detection within 10 minutes at room temperature,and real-time fluorescence RPA can complete the detection within 20minutes.The minimum detection concentration of the two methods for genomic DNA of N.seriolae is 100 pg/μL,and the minimum detection limit for recombinant plasmid p MD18-ITS is 10~3copies/μL.The sensitivity is consistent with PCR and has high specificity.In addition,we conducted a toxicity test on largemouth bass with N.seriolae to monitor the earliest time point at which the pathogenic bacteria could be detected.The results showed that the detection method we established can detect pathogenic bacteria 7 days before the onset and death of largemouth bass(Micropterus salmoides),and can achieve early diagnosis of nocardiosis.Finally,we evaluated the clinical applicability of the RPA-LFD and real-time fluorescence RPA detection methods for N.seriolae.Test results for clinical samples were consistent with PCR,confirming the strong clinical applicability of the method we have established.2.Establishment of a visual and rapid detection method for S.agalactiae by RPA-LFD and real-time fluorescence RPABased on the Sip gene sequence of S.agalactiae,we have established a visual rapid detection method for S.agalactiae and real-time fluorescence RPA.Similarly,we conducted experiments to screen the optimal reaction conditions,verify the specificity,and perform sensitivity testing,early diagnosis,and clinical utility evaluation for this method.The RPA-LFD detection method can achieve rapid detection within 15 minutes at room temperature,and the real-time fluorescence RPA detection method can complete the detection within 20 minutes.The minimum detection limit for recombinant plasmid p MD18-Sip is 10~4copies/μL,and the minimum detection concentration for genomic DNA of S.agalactiae is 10pg/μL,consistent with PCR sensitivity.Similarly,five days before the onset and death of tilapia(Oreochromis mossambicus),the method we established and the pathogen can be detected,which can make early diagnosis of S.agalactiae disease.The detection results of clinical samples are consistent with PCR,and have strong clinical practicality.In summary,we have established a convenient and sensitive visual rapid diagnostic method for the pathogenic bacteria of N.seriolae and S.agalactiae in fish,laying an important product foundation for the development of on-site rapid diagnostic reagents for N.seriolae and S.agalactiae in fish.