PCR conditions were 94°C for 3 min, followed by 40 cycles of DNA amplification
(45 s at AZD2281 chemical structure 94°C, 1 min at 61°C, and 1 min 30 s at 72°C) and 8 min incubation at 72°C. PCR products were analyzed on 1.2% (w/v) agarose gels by electrophoresis at a constant voltage (2 V/cm). The non-structural protein gene nsP3, the capsid proteins genes and 3’-UTR sequences were cloned and sequenced. Sequence analysis and phylogenetic comparisons Sequence data were analyzed using computer programs such as DNAMAN and DNASTAR. Phylogenetic analyses were performed by the neighbor-joining method using MEGA (version 5.05; http://www.megasoftware.net/). Previously published GETV sequences used in this study include sequences YN08 isolates MM2021 (from Malaysia, GenBank:AF339484), MAG (from Russia, EF631998), ALPV_M1 x(from China, EF011023), GETV_M1 (from China Hainan, EU015061), MPR (MPR from Mongolia, EF631999), S_KOREA (from South Korea, AY702913), HB0234(from China Hebei, EU015062), YN0540 (from China Yunan, EU015063), and SAGV (Sagiyama virus from Japan, AB032553). Acknowledgments We thank Ms. Ming Adriamycin order Qing for her administrative assistance. This work was financially sponsored by the key selleck compound program (no. U1036601 ) and the youth fund program(no. 81101618) from the National Natural Science
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