Application Of RPA In The Detection Of The Aquaculture Viral Pathogens And Epidemic Investigation Of Herpes Virus In Scapharca Subcrenata

Abalone Herpesvirus(AbHV),Redspotted grouper nervous necrosis virus(RGNNV)and Ostreid herpesvirus(OsHV-1)are three serious viruses that infect animal populations in aquaculture.There are currently no effective treatments for any of these three viral diseases.The preven-tion and control of aquaculture diseases is"in case of priority".At home and abroad,in terms of the diagnostic technology of aquatic diseases,the various detection techniques have been established,such as PCR,qPCR,LAMP,antigen-antibody detection and nucleic acid hybridization detec-tion.In this study,the suitable primers and probes were designed for each of the above three viruses,and real-time quantitative polymerase amplifica-tion(qRPA)was used for viral detection.The sensitivity and specificity of detection were evaluated by comparison with detection by quantitative PCR(qPCR).The results showed that the sensitivity of AbHV detected by qRPA was 100 copies,and the reaction time was only 20±0.50minutes.The detection percentage of samples infected with AbHV were96%and 93%by qRPA and qPCR,respectively.The sensitivity RGNNV detected by qRPA was also 100 copies.The detection percentage of RGNNV by qRPA and qPCR were 97%and 95%,respectively.For the detection of OsHV-1,one set of specific primer and probe designed using ORF95 as a template can detect 5 copies of DNA,which was much higher than the sensitivity of the qPCR established in this experiment(100 copies).For clinical samples,the percentage of viral load sample detected by the two methods was 22%.In addition,for the verification of the different samples infected with the three viruses,data showed that there was a strong correlation between qRPA and qPCR detection(R~2>0.8).Whatman FTA card is a filter device that can collect,transport,archive and purify all kinds of biological samples at room temperature.This study used this FTA card to extract RNA from the brain tissue of the grouper infected with RGNNV.The obtained RNA can be directly used in the PCR experiment and successfully detected RGNNV.This method greatly simplified the process of virus extraction,saving time and cost to a large extent.In order to visualize the results,we tried to detect AbHV,RGNNV and OsHV-1 by using RPA combined with lateral flow dipstick(LFD),and verify the sensitivity of the method.After the positive plasmids of the three viruses were incubated for 5 min,their detection limits of LFD were:10 copies,10 copies and 100 copies.In this study,qPCR was used to detect the Scapharca subcrenata infected with OsHV-1 in 15 coastal cities.We had a deep understanding of the geographical distribution and infection rate of OsHV-1 in Scapharca subcrenata.Results of the epidemiological survey showed that the virus was not detected in other areas except for Shenzhen,Dalian,Lianyungang and Nantong.At the same time,it was also found that OsHV-1 can be detected in Scapharca subcrenata collected in Shenzhen during the months of April,August and September,and the highest level of virus was detected in August.This coincided with the prevalence of OsHV-1,which was easily extinguished at water temperatures of 28~30°C.