Strategy for detecting low concentrations of genomic DNA in environmental DNA assessments in the Brazilian Amazon

This study employed a pre-amplification step using specific primers flanking a mini-barcode (105 bp) within the mitochondrial COI gene in Prochilodus nigricans to improve detection in environmental DNA (eDNA) from water samples collected in Amazonian floodplain lakes. Detecting rare and economically important species through eDNA analysis can be challenging due to issues such as cross-amplification and false negatives in qPCR. The addition of Pre-amplification solved these problems, especially for P. nigricans. Without the pre-amplification step, specific targets were not detected by qPCR; however, Cq values ranged from 35.96 to 39.67 in the pre-amplified samples, except for in one of the sampled lakes. A linear and positive correlation between gDNA dilution and Cq values was observed, demonstrating improved qPCR performance with pre-amplification. In summary, pre-amplification is presented as a robust approach to enhance the sensitivity of detecting specific targets in eDNA from Amazonian lake ecosystems.