Detection of Bovine Group A Rotavirus Using Rapid Antigen Detection Kits, RT-PCR and Next-Generation DNA Sequencing

MINAMI-FUKUDA, Fujiko; NAGAI, Makoto; TAKAI, Hikaru; MURAKAMI, Toshiaki; OZAWA, Tadashi; TSUCHIAKA, Shinobu; OKAZAKI, Sachiko; KATAYAMA, Yukie; OBA, Mami; NISHIURA, Naomi; SASSA, Yukiko; OMATSU, Tsutomu; FURUYA, Tetsuya; KOYAMA, Satoshi; SHIRAI, Junsuke; TSUNEMITSU, Hiroshi; FUJII, Yoshiki; KATAYAMA, Kazuhiko; MIZUTANI, Tetsuya

Five immunochromatographic assays, the Dipstick 'Eiken' Rota (Eiken Chemical Co., Ltd., Tokyo, Japan), RapidTest Rota Adeno (Sekisui Medical Co., Ltd., Tokyo, Japan), BD Rota/Adeno Examan stick (Becton, Dickinson and Franklin Lakes, NJ, U.S.A.), Rapid-SP "Rota" (DS Pharma Biomedical Co., Ltd., Osaka, Japan) and Immuno-Card ST Rotavirus (TFB, Inc., Tokyo, Japan), and two latex agglutination assays (Rotalex Dry (Sekisui Medical Co., Ltd.) and Rotascreen (Denka Seiken Co., Ltd., Tokyo, Japan) were conducted in this study. Serial 10-fold dilutions of IS1, NCDV and KK3 were prepared in phosphate buffer saline (PBS), and 100 µl of viral dilutions were mixed with the extraction solution provided with each kit. The procedures were conducted according to the manufacturer's instructions for each kit. For RT-PCR, 100 µl was used to extract viral RNA from each sample using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instrutions. All extracted RNAs were denatured at 97°C for 3 min and immediately placed on ice. Then, one-step RT-PCR was performed with PrimeScript One Step RT-PCR Kit Ver. 2 (Takara, Otsu, Japan) using the extracted RNA described above. The primer pair Beg9/End9 [4] was used for amplification of the full-length VP7 under the following conditions: 50°C for 30 min and 95°C for 15 min, followed by 35 cycles of 94°C for 1 min, 45°C for 1 min and 72°C for 1 min with a final extension of 72°C for 10 min. The primer pair BRVF/BRVR described by Zhu et al. (2010) [21] was used for amplification of partial VP6 gene under the following conditions: 50°C for 30 min and 94°C for 5 min, followed by 35 cycles of 94°C for 50 sec, 55°C for 50 sec and 72°C for 1 min with a final elongation of 72°C for 10 min. The RT-PCR products were electrophoresed on 2% agarose gel.