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

The results regarding the detection limits of RAD kits and RT-PCR for the three bovine RVA strains are shown in Table 1 . Limited dilution analyses showed that the Dipstick exhibited the highest sensitivity to all strains of bovine RVA of the seven RAD kits. RT-PCR using primer pairs Beg9/End9 and BRVF/BRVR had 10 0 -10 3 and 10 2 -10 3 -fold higher sensitivity than the Dipstick, respectively. Furthermore, limited dilution analyses using two fecal samples were performed and revealed that RT-PCR using primer pairs Beg9/End9 and BRVF/BRVR had 10 1 -10 2 and 10 1 -10 3 -fold higher sensitivity than the Dipstick, respectively (data not shown). For NGS, we performed RNA-seq using commercial kits. First, total RNA was prepared from fecal samples diluted 1:9 (W/V) in sterile PBS using ISGEN LS (Nippon Gene, Tokyo, Japan), and this was followed by DNase I treatment (Takara, Otsu, Japan). Quantification of the RNA samples was performed using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, U.S.A.). RNA samples were normalized to 10-100 ng of RNA, and RNA library preparation was performed using a NEBNext ® Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, U.S.A.) according to the manufacturer's intruction. Briefly, RNA was fragmented, and then double-stranded cDNA was synthesized. The resulting double-stranded cDNA was end-repaired before ligation of Illumina-specific adaptors and size selection of the libraries with approximately 200 bp inserts and was finally PCR enriched. After assessing the library quality on a Bioanalyzer ® (Agilent Technologies, Santa Clara, CA, U.S.A.), sequencing was carried out on a MiSeq sequencer (Illumina, San Diego, CA, U.S.A.) using 51 single-end reads. Data analysis was performed using the MiSeq Reporter Software (Illumina) to generate sequence data in FASTQ format. To obtain consensus sequences for 11 segments of bovine RVA, respectively, reads were aligned with the sequences of all 11 segments (G6-P [5]-I2-R2-C2-M2-A3-N2-T6-E2-H3) of the bovine RVA strain WC3 [1, 15] as reference sequences using CLC Genomics Workbench (CLC bio, Cambridge, MA, U.S.A). For VP4, VP7 and NSP1, the sequences of NCDV Lincoln (P [1] ), B223 (P [11] ) [17] , A5 (G8) [16] , 61A (G11) [15] and B383 (A13) [8] were also used as references.