Establishment And Preliminary Application For Quantitative Real-time RT-PCR Method Of Small Ruminant Lentiviruses

The caprine arthritis-encephalitis virus and maedi visna virus are collectively referred to as small ruminant lentiviruses,which is the mainly cause of chronic persistent infection in goats and sheep.It is characterized by arthritis and encephalomyelitis in goats,interstitial pneumonia in sheep and so on.These two kinds of virus infection have certain host specificity,that is,the CAEV is more likely to infect goats,while MVV tends to infect sheep.But in recent years,the molecular epidemiology has found that it can cross species infection.Based on the pol-gag gene sequence for viruses,it was divided into five subgroups,which include A,B,C,D,E.The currently established methods for biological detection of pathogenic molecules are only applicable to the detection of strains of subpopulations of some genes.Therefore,it is very necessary to establish a universal detection method for CAEV and MVV nucleic acid.In this study,to compare 31 strains of small ruminant lentivirus proviral genome sequences which published in Gen Bank.A sequence about 150bp in vif gene was selected as diagnostic marker and degenerate primers were designed.It was obtained by whole gene synthesis as a positive control template for PCR reaction,and a general PCR detection method for small ruminant lentivirus was established.On this basis,the real-time fluorescence quantitative RT-PCR method was established by using SYBR Green I dye.The animal experiment of CAEV artificial infection was carried out.In order to verify the establishment of pathogenic biological detection methods,the anticoagulant blood was collected for 5mL at different times.Which is,on the 70th and 10th days before infection.On the 4th,8th,15th,23th,28th,42th,58th,71st,84th,99th and 113th days after infection.The blood samples were processed within 72 hours after each blood collection,to extract total DNA from 1mL whole blood and detect the pre-viral DNA of CAEV.After centrifugation of anticoagulated blood,200?L of plasma was extracted viral RNA and reverse transcribed into cDNA.Then,the pathogen and its load were detected by using PCR and real-time fluorescence quantitative RT-PCR.Meanwhile,we used IDEXX SRLV antibody screening kit to detect serum antibodies.In addition,a total of 165 goat samples from 2017 to 2018 in Xinjiang Urumqi,Aksu,Wusu,Bachu and Fuyun counties were collected for antibody detection and etiological detection.The results of the study indicate that the 150bp sequence located on the SRLV vif gene can be used as its universal diagnostic marker.The established universal PCR assay has a sensitivity of 10~3 molecular copies.It was initially confirmed that can be used for the detection of RNA and proviral DNA of wild-type strains of CAEV and MVV.The sensitivity of real-time fluorescent quantitative RT-PCR method established by SYBR Green I dye was not less than 100 copies,the linear range is 10~2-10~7 copies of the molecule,and it can be used for detection of CAEV animal infection,which was earlier than the time when the infected antibody was detected.In addition,25%and 20%CAEV serological positive samples were detected in Wusu county and Bachu county respectively.The serological positive rate of CAEV in Xinjiang is 2.4%.It is confirmed again that Xinjiang has the disease epidemic.The method established in this study is expected to be used for etiological detection of SRLV in China.It is necessary to carry out a large-scale CAEV epidemiological investigation in order to grasp its epidemic situation and formulate prevention and control programs in Xinjiang.