Fig 1.
(A) qPCR results using the first S-Creek DNA extraction (0 ft) as template with the CytB104 primers/probe, the HB196 primers without the first step enrichment, and the HB196 primers using HB503-enriched PCR product as template (B) qPCR standard curves for CytB104, HB196, HB196 with 20 cycles of HB503 template enrichment, HB196 with 35 cycles of HB503 template enrichment. Template concentrations range from 500,000 copies/µL to 5 copies/ µL (Error bars represent standard error, n = 3).
Fig 2.
ddPCR mtDNA amplicon concentration as a function of distance.
(A) DNA schematic of the HB primer sets [67] (B) ddPCR quantification results for each sample collected along S-Creek in November of 2017* (Blue trendline: exponential decay equation fit using Levenberg–Marquardt algorithm) (C) Satellite view of S-Creek and each sampling location** Credit: U.S. Geological Survey (D) PCR product from HB196 primers on HB503 enriched S-Creek eDNA extractions (Lanes 1: 0 m, Lane 2: 245 m, Lane 3: Positive control, Lane 4: NTC) and CytB104 primer on S-Creek eDNA extractions (Lanes 5: 0 m, Lane 6: 245 m, Lane 7:Positive control, Lane 8: NTC). * Error bars represent standard error, n = 5. **The weeks following the sampling event at S-Creek in 2017 brought heavy rains and flooding and the detected hellbender populations were confirmed to no longer reside there.
Fig 3.
PCR effeciency for CytB104 primers in the presence of self-generated off-target amplicon.
(A) Ct-value results from qPCRs conducted with three different ratios of Target: Non-Target DNA* (B) PCR results using the CytB104 primer set on 4 N. maculosus DNA templates (Lanes 1-4, No Template Controls Lanes 5-6). ~ 250 bp band in lane 3 was gel-purified and used as off-target template for qPCR analysis displayed in A (C) NCBI BLASTn results for the ~ 100 bp product gel-purified and Sanger sequenced from CytB104 PCR (Fig 3B, Lane 1) indicating exact homology with the N. maculosus cytochrome-B gene. *Error bars represent standard error, n = 3.
Table 1.
Primer sequence comparison.