Serotype identification of enteroviruses
Swabs were stored in a dedicated Universal Transport Medium (UTM) (Yocon, Beijing, China) for transport. Stool samples were diluted to a 10% suspension using MEMs. After thorough mixing, 200 μl of clinical samples were used to extract RNA using the QIAamp Viral RNA Mini Kit (Qiagen, CA) according to the manufacturer’s instructions. EV, EV-A71, CVA16, CVA6 and CVA10 were confirmed using commercial real-time RT-PCR Kits (BioPerfectus technologies, Jiangsu, China). Samples positive for universal EV but negative for the above serotypes were named as un-serotyped EV (UEV).
Serotyping of UEV
A reverse transcription-nested PCR was used for UEV serotyping. Simply, pan-enterovirus specific primer sets targeting the 5’UTR for nested RT-PCR to give a 389-bp-long product was as follows: the first round PCR using primers Out-F 5’-CYTTGTGCGCCTGTTTT-3’ and Out-R 5’-ATTGTCACCATAAGCAGCC-3’ (530bp), then the second round PCR using primers Inner-F 5’-CAAGYACTTCTGTMWCCCC-3 and Inner-R 5’-CCCAAAGTAGTCGGTTCC-3’(389bp) (16). For the first RT-PCR, PrimeScript One Step RT-PCR Kit Ver.2 (Takara) was used with the following reaction conditions: 50℃ reverse transcription for 30 min, 94℃ pre-denaturation for 2 min, 40 cycles of 94 ℃ 15 s, 55 ℃ 30 s, 72℃ 1 min. For the second PCR, TaKaRa Taq™ HS Perfect Mix (Takara) was used according to the manufacturer’s protocol. The final PCR products were subjected to 2.0% agarose gel electrophoresis for analysis. PCR products of the second PCR were purified using the QIAquick PCR Purification Kit (Qiagen), and then amplicons were sequenced in both directions using the ABI 3730 Genetic Analyser (Applied Biosystems).