Establishment And Application Of Rapid Detection For Infectious Spleen And Kidney Necrosis Virus And Edwardsiella Ictaluri By Recombinase Polymerase Amplification (RPA)

Mandarin fish(Siniperca chuatsi)and yellow catfish(Pelteobagrus fulvidraco)are two important freshwater farmed fishes with excellent meat quality and high economic value,have been widely cultured in China.In recent years,with the rapid development of intensive culture,the infectious diseases caused by infectious spleen and kidney necrosis virus(ISKNV)and Edwardsiella ictaluri have constantly broken out,which cause serious economic losses to the mandarin fish and yellow catfish farming industry in China.In order to effectively control the outbreak and spread of the two infectious diseases,rapid and sensitive detection of ISKNV and E.ictaluri is necessary.Therefore,we hope to establish a rapid detection method,which can detect ISKNV and E.ictaluri in the field.In this study,we used the RPA technique for the detection of ISKNV and E.ictaluri,and established the RPA rapid detection method for ISKNV and E.ictaluri,in order to achieve early diagnosis of the two pathogens and effectively control the outbreak and spread of the two infectious diseases.This study is divided into two parts:the establishment of the RPA rapid detection method for ISKNV and the establishment of the RPA rapid detection method for E.ictaluri.1.The establishment of ISKNV RPA rapid detection method.We designed 6 sets of RPA primers for the MCP and ORF007 genes of ISKNV,and the primers MCPF181/MCPR182 and 007F197/007R198 were selected as the most suitable primers for subsequent ISKNV RPA detection.Then we optimized the reaction conditions of ISKNV RPA and RPA-LFD detection methods.We found that 38°C was the optimal reaction temperature for the ISKNV RPA and RPA-LFD methods and 30 min was the optimal reaction time for the ISKNV RPA and RPA-LFD methods.The optimal conditions were used for subsequent ISKNV RPA and RPA-LFD tests.We used nucleic acid samples of different pathogens as templates and used ISKNV RPA and RPA-LFD methods for detection.And we found that only the detection result of ISKNV DNA was positive.We used gradient dilute plasmids as templates and used ISKNV RPA and RPA-LFD methods for detection.We found that the lowest detection limits of ISKNV RPA and RPA-LFD methods were both 10~2 copies/μl.We used the DNA of mandarin fish as the clinical samples and used RPA,RPA-LFD and PCR methods for detection.We found that the ISKNV RPA and RPA-LFD methods showed the same positive rate as the PCR method.In short,the ISKNV RPA and RPA-LFD detection methods could accomplish the detection at 38°C for30 minutes.The specificity of these methods was high,and the sensitivity of these methods was 10 times higher than that of the PCR method.And these methods showed the same effect as PCR in the detection of clinical samples.2.The establishment of E.ictaluri RPA rapid detection method.We designed 9 sets of RPA primer combinations for the serC gene of E.ictaluri,and the primers serCF564/serCR569 had the best amplification effect and were selected as the most suitable primers for the subsequent RPA detection of E.ictaluri.Then we optimized the reaction conditions of E.ictaluri RPA and RPA-LFD detection methods.We found that38°C was the optimal reaction temperature for the E.ictaluri RPA and RPA-LFD methods and 30 min was the optimal reaction time for the E.ictaluri RPA and RPA-LFD methods.The optimal condition was used for subsequent E.ictaluri RPA and RPA-LFD tests.We used the nucleic acid samples of different pathogens as templates and used the E.ictaluri RPA,RPA-LFD and real-time RPA methods for detection.And we found that only the detection result of E.ictaluri DNA was positive.We used gradient dilute plasmids and genomic DNA as templates and used the E.ictaluri RPA,RPA-LFD and real-time RPA methods for detection.we found that the lowest detectable concentration of plasmids was10~2 copies/μl by the E.ictaluri RPA,RPA-LFD and real-time RPA methods,and the lowest detectable concentration of genomic DNA was 1 pg/μl by the E.ictaluri RPA,RPA-LFD and real-time RPA methods.We used the DNA of yellow catfish as the clinical samples and used RPA,RPA-LFD,real-time RPA and PCR methods for detection.We found that the E.ictaluri RPA,RPA-LFD and real-time RPA methods showed the same positive rate as the PCR method.In short,the E.ictaluri RPA,RPA-LFD and real-time RPA detection methods could accomplish the detection at 38°C for 30 minutes.The specificity of these methods was high,and the sensitivity of these methods was the same as that of the q RT-PCR method and 10 times higher than that of the PCR method.And these methods showed the same effect as PCR in the detection of clinical samples.The results of this study showed that the RPA detection methods developed for ISKNV and E.ictaluri were very suitable for the rapid detection of ISKNV and E.ictaluri.And the RPA-LFD detection methods of ISKNV and E.ictaluri had great application potential for the detection in the field because the methods were easy to operate and didn’t need complicated equipment.