The Establishment Of RT-LAMP Assay To Detect BVDV And The Preliminary Development Of Inactivated Brv And BVDV Vaccine

Bovine rotavirus belongs to Reoviridae family, Rotavirus genus. And Bovine viraldiarrhea virus belongs to Flaviviridae family, Pestivirus genus. They are both majorpathogens of calf viral diarrhea. BVDV cause reproductive disturbance, Mucosal diseaseand persistent infection. Recent years, the disease spreads across the world and has causedgreat harm.In this study, two Baoding local strains of Bovine viral diarrhea virus were isolatedfrom the samples of suspicious cases, and named as BD-2, BD-4respectively. BD-2andBD-4were cultured on MDBK, from F1to F20. After DNA sequencing and TCID50of thetwo strains, BD-2was chosen as a vaccine strain.At the same time,4of RT-LAMP primers of the sequence coding protein gP48wasdesigned by using Primer Explorer4.0，aiming at Establish a rapid method for detection ofBVDV by RT-LAMP. The reaction conditions, reaction time and reaction temperature wereoptimized. The specificity, the sensitivity were tested as well. The experiments resultsshowed that the optimal reaction time and temperature were determined to be40minutes at56℃. RT-LAMP turned out to be more sensitive and timesaving than conventionalRT-PCR. Determination of PT-LAMP result needs no expensive equipment. PT-LAMP alsoshowed advantages on specificity, sensitivity and simplicity, which was suitable for rapiddetection of BVDV in clinic.Bovine rotavirus strain RV+was cultured on Marc-145, and also identified by DNAsequencing and TCID50. RV+strain showed great stability. So it can be used as a vaccinestrain. BD-2F5and RV+F1were used to make inactivated BRV and BVDV vaccine.Then，the safety and Immune efficacy were test on Kunming mice: results showed that theinactivated BRV and BVDV vaccine was safe and the protection rate reach as93.3%.