Agarose gel purification kit

The cytochrome b (Cyt b) gene was amplified using polymerase chain reaction (PCR) with primers L14742 and H15915 (Xiao, Zhang, & Liu, 2001). Each 50 μl PCR contained 1–10 ng of template DNA, 5 μl of 10 × PCR buffer, 1 μl of dNTP mix (10 mM), 10 pmol of each primer, and 2 U of rTaq polymerase (TaKaRa). PCRs were conducted in a thermal cycler (T100; Bio‐Rad) with the following program: one cycle of denaturation at 95°C for 4 min; 30 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 90 s; and one final cycle at 72°C for 10 min. Successful PCR products were separated by electrophoresis on a 1.0% agarose gel and purified using the Agarose Gel Purification Kit (Qiagen).
Several samples with distinct phylogenetic clades were also used to test the existence of cryptic species using COI gene. The primers and PCR information were as Ward, Zemlak, Innes, Last, and Hebert (2005).
Purified products then were sequenced with an ABI PRISM 3730 sequencer. Sequencing primers were the same as those used for PCR amplification. All unique sequences have been deposited in GenBank.

Positive individual samples were subjected to nested RT-PCR reaction as described earlier^{26 (link),27 (link)}. In brief, a ~ 341 bp fragment of the HEV RNA dependent RNA polymerase (RdRp) gene was amplified and the resulting PCR product was purified using the agarose gel purification kit (Qiagen, Germany) and subjected to direct sequencing using the BigDye dideoxy termination kit on the ABI 3500 sequence analyzer (Applied Biosystems, Germany). The full genome sequencing was performed on two positive cases one imported and one domestic case. The full genome sequence was performed according to Lee et al.^{22 (link)}. The full HEV genome was amplified in 19 fragments and the PCR products were purified from agarose gel and sequenced using the same protocol as for the RdRp fragments. The fragments were assembled using the assembly tool of the Geneious prime software (version 2021.12.2)^{28 (link)}. The generated sequences were multiple aligned using Geneious software and phylogenetic trees were produced using the maximum likelihood method with 1000 bootstrap replicates. Sequences were deposited in Genbank and were assigned the accession numbers MZ027568–MZ027584 for the RdRp gene sequences and MW835252–MW835253 for the two full genome sequences.