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).