Establishment And Application Of A Recombinase-aided Amplifica Tion Assay For Detection Of Avian Reticuloendotheliosis Virus

Avian reticuloendotheliosis（RE）is a neoplastic infectious disease caused by Reticuloendotheliosis virus（REV）,which mainly invades the lymphatic system of poultry and causes serious immunosupprcssion.In recent years,epidemiological investigation and research have revealed that REV infection has become quite common in chicken flocks in many countries and in China.At the same time,REV contamination in vaccines has been reported,making REV surveillance and decontamination extremely important for the prevention and control of the disease.,REV can be transmitted vertically through seed eggs and is one of the viruses that need to be decontaminated from seed sources,while SPF chicken embryos carrying REV could lead to REV in the attenuated vaccine.Virus isolation by cell inoculation is one of the standard methods for detecting REV.Although this method is accurate,it also has disadvantages.On the one hand,this method is time-consuming,and on the other hand,it requires high level of human operation ability and testing conditions.At present,a variety of molecular biological methods have been applied to the detection of REV,such as RT-PCR and fluorescence quantitative RT-PCR,butthey also have high requirements for personnel operation level,detection instruments and operation environment,and are not suitable for promotion in enterprises.It is urgent to establish a rapid nucleic acid detection method for REV in enterprises.In this study,a fluorescent RAA assay for the detection of REV nucleic acid was established based on recombinant enzyme-mediated isothermal amplification（RAA）.Four pairs of primers and probes were designed for the conserved region of the pol gene based on the GenBank genome sequence of REV,and the best primers and probes were selected by gel RAA.The reaction system and reaction conditions were explored,and their sensitivity,specificity and repeatability were compared experimentally.The results show that the sensitivity of the fluorescence RAA assay established in this study was 102 copies/μL,which is comparable to that of TaqMan fluorescent PCR and 10 times more sensitive than ordinary PCR.This assay was only able to detect REV nucleic acid,and did not show any crossreactivity with chicken Marek’s disease virus,avian leukemia virus,avian infectious anemia virus,avian hepatitis E virus and avian adenovirus.This study further explored the practicability of this assay in different situations.Firstly,different doses of REV were added into avian attenuated vaccine to artificially simulate REV contamination in vaccines,and three assay were used to detect REV contamination.The results showed that the fluorescence RAA assay could detect the REV contamination at a minimum of 1 TCID50,the TaqMan real-time PCR method could also detect 1 TCID50,while the ordinary PCR could only detect REV contamination at 103 TCID50.Then,REV was inoculated into SPF chicken embryos and swab samples were collected and detected by different assay.The results showed that the sensitivity of fluorescence RAA were completely consistent with that of TaqMan real-time PCR.Finally,the suspected clinical samples were tested by the fluorescence RAA assay.It showed that the detection results of RAA assay was consistent with virus isolation method.Lastly,fluorescence RAA was to detect the suspected REV positive samples sent for inspection in various regions.Two REV positive samples were identified from 15 samples,which was consistent with the virus isolation and identification results;Furthermore,the two isolated REV gp90 genes strains were further sequenced and analyzed,and the homology of the two strains of REV gp90 was found to be significantly different..In conclusion,the fluorescence RAA assay for REV detection based on recombinant enzyme mediated isothermal nucleic acid amplification was established in this study.This assay has the advantages of high sensitivitygood specificity,simple operation,convenience and high accuracy.This assay provided a strong technical support for the rapid detection of REV on site and the detection of REV contamination in avian attenuated vaccine.