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Detection of flaviviruses

HH Hayato Harima
YO Yasuko Orba
ST Shiho Torii
YQ Yongjin Qiu
MK Masahiro Kajihara
YE Yoshiki Eto
NM Naoya Matsuta
BH Bernard M. Hang’ombe
YE Yuki Eshita
KU Kentaro Uemura
KM Keita Matsuno
MS Michihito Sasaki
KY Kentaro Yoshii
RN Ryo Nakao
WH William W. Hall
AT Ayato Takada
TA Takashi Abe
MW Michael T. Wolfinger
MS Martin Simuunza
HS Hirofumi Sawa
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Tick RNA samples were examined to detect flavivirus via RT-PCR using a One Step RT-PCR Kit v2 (Takara, Shiga, Japan) with pan-flavivirus primer set (see Supplementary Table S2 online) based on the conserved sequence within the flavivirus NS5 protein as previously described54. The RT-PCR conditions were as follows: initial reverse transcription step at 50 °C for 30 min; PCR activation step at 94 °C for 2 min; 43 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. PCR products were subjected to direct sequencing using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).

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