Isolation And Identification Of Bovine Rotavirus And Development Of Detection By A RT-LAMP Method

Bovine rotavirus belongs to Reoviridae family, Rotavirus genus. It is major pathogen of calf viral diarrhea. The disease spreads across the world and has caused great financial losses. RT-PCR and ELISA, as commonly used detection methods, are hardly universal in clinic diagnosis due to the need for expensive equipment and professional researchers. So a convenient and simple detection method is desperately in need. Loop mediated isothermal amplification (LAMP) technique is a novel nucleic acid amplification method which relies on the Bst DNA polymerase to accomplish auto cycling strand displacement DNA synthesis. It is an isothermal reaction at a constant temperature and the result can be directly judged through the fluorescent staining by SYBR GREENⅠor judged from the result of centrifugal precipitate. Therefore, LAMP is more convenient and faster than ELISA and RT-PCR for detecting BRV.A Suspected BRV strain was isolated from the clinical feces samples originated from cattle farms in Baoding, then cultured the BRV strain on Marc-145 cells at 37℃in CO2 incubator. Cytopathogenic effects (CPE) (circle shrinking, disintegration) were showing after four generations of blind passages. According to the published BRV VP7 gene sequence on GenBank, a set of special primers of the VP7 gene were designed to detect the cell culture fluid in which Marc-145 showed CPE, and the cDNA of the isolates VP7 gene were amplified by RT-PCR. The result showed that the product size was 342bp, which was the same length as we expected. The RT-PCR product was purified and recovered. Transformed clone and sequence determination showed that test results were in accordance with expected results. Results of physicochemical characteristic tests indicated that the BRV strain was not able to endure heat and was not sensitive to chloroform, strong acid and diethyl ether either. This study successfully isolated bovine rotavirus, and named this strain as BD.The study detected BRV by using RT-LAMP technology. According to published BRV VP7 gene sequence on GenBank, a set of primers, which composed of outer primers, inner primers, was designed by using Primer Explorer4.0. BRV was detected by RT-LAMP using the primers, and the reaction conditions were optimized. The experiments results showed that the optimal reaction time and temperature were determined to be 50 min at 62℃. Every component concentration after optimization was Mg2+ 2.5μL,dNTPs 3.5μL,Bst DNA polymerase 1.5μL and optimum concentration ratio between inner and outer primers was 1:6. RT-LAMP turned out to be more sensitive than conventional PCR. Its sensitivity was 100 times as sensitive as RT-PCR. No cross reaction was observed from the samples and other related viruses. Testing results among groups and within the group were the same. It demonstrated that this detection method was of good stability and repeatability. Forty-nine samples were detected by RT-PCR and RT-LAMP respectively in this study. And the test results of the two detection methods were identical. The method of using RT-LAMP method to detect BRV was successfully established.