| Maedi-Visna disease(MVD)is a progressive multi-organ inflammatory disease of sheep caused by Maedi-Visna virus(MVV);Ovine Pulmonary Adenocarcinoma(OPA)is a contact lung tumor disease caused by Jaagsiekte Sheep Retrovirus(JSRV).Cases of mixed infections have been reported in different countries and regions,and studies have found that JSRV epidemic conditions accelerate MVV transmission,and there are no effective control measures for both.Early detection and isolation is the most feasible way to avoid widespread transmission,so it is especially important to establish a rapid detection method to control the spread of the disease and reduce economic losses.Recombinase Polymerase Amplification(RPA)is fast and does not require expensive instrumentation and is widely used for the detection of various pathogens.In this experiment,we designed fluorescent RPA primers and probes based on the conserved sequence of MVV gag gene on Genbank for rapid detection of MVV in diseased sheep,and established a real-time fluorescent RPA assay for MVV-gag.The optimal reaction temperature was determined to be 39℃,and the specificity test showed no cross-reactivity with other pathogens prone to respiratory symptoms;the minimum detection limit was 100 fg/μL,which was more sensitive than gag-PCR;eight clinical samples kept in the laboratory were tested,and the normal PCR test was used as a control test with the same results.Meanwhile,in order to specifically detect the Inner Mongolia MVV strain and establish a more sensitive real-time fluorescent RPA assay,the primers and probes were designed based on the MVV LTR gene sequence,and a real-time fluorescent RPA assay for MVV-LTR was established.The optimal reaction temperature was determined to be 41°C and the minimum detection limit was 10 fg/μL.The method has good sensitivity,reproducibility and specificity and was performed on 8 clinical samples kept in the laboratory,and the normal PCR assay was used as a control test,and the results were as expected.In order to rapidly detect mixed cases of JSRV and MVV infection,this experiment established a dual real-time fluorescent RPA assay for JSRV-env and MVV-LTR based on the MVV-LTR fluorescent RPA assay and the JSRV fluorescent RPA assay.The optimal reaction temperature was determined to be 42°C,the minimum detection limit was 1 ng/μL,the sensitivity was consistent with the dual PCR,and it was able to specifically identify mixed cases of infection.8 clinical samples kept in the laboratory were tested,and the dual PCR assay was used as a control test,and the results were consistent.In summary,this experiment successfully established the MVV-gag real-time fluorescent RPA detection method,MVV-LTR real-time fluorescent RPA detection method and JSRV-env and MVV-LTR dual real-time fluorescent RPA detection method,which provide technical support for rapid diagnosis of MVD and mixed infection cases and effectively control the widespread spread of the epidemic. |
