| Popis: |
Here we describe GoFish, a streamlined environmental DNA (eDNA) presence/absence assay. The assay amplifies a 12S segment with broad-range vertebrate primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens representing 36 northwestern Atlantic taxa not well documented in GenBank. Using the newly obtained and published 12S records, we designed GoFish assays for 11 bony fish species common in the lower Hudson River estuary and tested seasonal abundance and habitat preference at two sites. Additional assays detected nine cartilaginous fish species and a marine mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. The assay can be carried out from water collection to result in three days. The main limitations so far are species with shared target sequences and inconsistent amplification of rarer eDNAs. We think this approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings. |
