Author: Niccolo Alfano; Anisha Dayaram; Jan Axtner; Kyriakos Tsangaras; Marie-Louise Kampmann; Azlan Mohamed; Seth Timothy Wong; M. Thomas P. Gilbert; Andreas Wilting; Alex Daivd Greenwood
Title: Non-invasive surveys of mammalian viruses using environmental DNA Document date: 2020_3_29
ID: nil1vv6h_46
Snippet: The primers listed in Suppl. Tab. 2 were designed to confirm by PCR the viral contig sequences generated by the three approaches (A, B, C see above) from the leech samples. For PCRs targeting RNA viruses, 50 uL of extract were digested with rDNase I (Ambion) following the manufacturer's protocol. The DNAse-digested extracts were then purified using the RNeasy MinElute Cleanup Kit (Qiagen). RNA was reverse transcribed into cDNA using iScriptâ„¢ Re.....
Document: The primers listed in Suppl. Tab. 2 were designed to confirm by PCR the viral contig sequences generated by the three approaches (A, B, C see above) from the leech samples. For PCRs targeting RNA viruses, 50 uL of extract were digested with rDNase I (Ambion) following the manufacturer's protocol. The DNAse-digested extracts were then purified using the RNeasy MinElute Cleanup Kit (Qiagen). RNA was reverse transcribed into cDNA using iScriptâ„¢ Reverse Transcription Supermix (Bio Rad). Sediment and water samples that tested positive for EHV and JSRV were screened using a previously described pan-herpes PCR [47] and for JSRV [48] , respectively. The resulting amplicons were Sanger sequenced.
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