Establishment Of Two Detection Methods For Bovine Enterovirus Based On LAMP And RPA

Bovine Enterovirus(BEV)is a member of the enterovirus genus of micrornaviridae.BEV is a common sunclinical infection pathogen,however,it can cause diarrhea and respiratory diseases in cattle,accompanied by orchitis,abortion and stillbirth.vertical transmission and horizontal transmission could be observed in infected animals,which causes the farm endemic continuously,then leads to the decline of production and breeding.So far,there is no specific drug or vaccine for BEV prevention and treatment.In order to control this disease,the most important thing is continuous pathogen monitoring.The traditional detection methods as virus isolation,PCR amplification and immunofluorescence detection technologies have the disadvantages of high cost of time and harsh operation requirements,which restrict the detection and application in the field detections.In recent years,on account of the advantages of high sensitivity,high specificity,simple operation and low requirements for equipment the new detection method called isothermal amplification technology has been used in clinic more popular.Based on these information,this study intends to establish the detection methods of BEV by lamp and RPA,further provide a solid technical support for the disease control.The LAMP detection method was established as follow.The recombinant vector p MD18-T-BEV HN1817-vp1 was constructed by molecular biotechnology based on the sequence of BEV HN1817 strain isolated in our laboratory,and the plasmid was used as the template for the following experiment.Primer Explorer V5 primer design software was used to design lamp primers.According to the experimental requirements,four groups of lamp primers were designed and screened.The detection system of lamp was initially established.The optimal reaction conditions were at 63?for 45 min.The lowest detection concentration of the method is 5.58×10~3 copies/?L?The results of specific tests showed that the BEV lamp was no reaction to the templates of DNA/RNA of BVDV,BRV and BPIV-3;further the 94 bp production were obtained by double enzyme digestion of Set?and Hin1?,and the sequencing results showed consistent with the BEV.The conservative range of VP-1 was selected to design the primers for the RPA detection method establishment.In this part,a visual RPA detection method was successfully established by screening the primer ratio and other systems factors based on BEV HN1817.The RPA detection method can effectively amplify the target fragment in only 15 minutes at a constant temperature of 36.7°C,and the detection threshold is 1×10~2copies/?L,compared to the detection threshold(1×10~3 copies/?L)of the conventional PCR method the sensitivity of the RPA detection method established in this experiment is significantlylower than that of the conventional PCR method.In addition,the two methods of visual recombinase RPA and LAMP were used to detect t 16 suspected positive clinical bovine oral swabs collected from the southwestern Henan cattle farm.Meanwhile,PCR detection method was used for comparsion,and the sequence of positive amplicons were identified by sequencing.The results showed that the detection rate of RPA and lamp were 87.5%,the same as the detection rate of LAMP.In conclusion,two detection methods for bovine enterovirus were establised based on RPA and LAMP,further employment in the detections of clinical samples showed the excellent accuracy,and the lamp detection results can be judged by the color changes in reactions,which would avail the use in field detection and provides technical support for the prevention and control of BEV.