FishDNAIDs: DNA barcoding as a tool in the development and validation in silico and in vitro of detection systems to four species of Characiformes of commercial importance in the Brazilian Amazon

Abstract

Background: The COI mitochondrial gene has been chosen as the "DNA barcode in animals" and the large quantity of genetic information in public databanks gives solid support for the use of DNA barcoding as a promising tool for the development of a specific molecular detection system.

Methods and results: The present study aimed to develop a Specific Molecular Detection System (SMDS: FishDNAIDs) (primers and probe sets) for the following four target species: Prochilodus nigricans, Potamorhina altamazonica, Psectrogaster rutiloides and Triportheus angulatus, in qPCR assays. In silico and in vitro tests (using gDNA) were performed to test these sets. The database generated contained the cytochrome c oxidase subunit I (COI) nucleotide sequence for 183 specimens of Characiformes, distributed in 34 species representing eight families. In silico, primers designed for the target species amplified different species from the same genus, except for P. rutiloides, which amplified only the target species. In the in vitro test, using the SYBRGreen^tm and TaqMan® fluorescence systems, both sets detected the respective target species (P. nigricans, P. altamazonica, P. rutiloides and T. angulatus). In the qPCR assays using SYBRGreen^tm, species considered to be related were also detected, in addition to the target species, with the exception of P. amazonica and P. essequibensis (correlated to P. rutiloides). All target species were detected in the qPCR assays using the TaqMan® system; however, with the SMDS PALT, the target species P. altamazonica was detected with low C_T values (22.21 ± 0.17) as well as the correlates of P. latior and P. pristigaster, though with high C_T values (23.51 ± 0.19 and 30.21 ± 0.95). This assay uniquely identifies known adult tissue samples from all four species.

Conclusions: The primers and probe sets developed can act as powerful tools for detecting the target Characiformes species.

Keywords: Conservation; DNA barcode; Detection; Monitoring; Sustainable development goals.