Condições de uso do eDNA (environmental DNA) no monitoramento de Prochilodus argenteus

Abstract

The environmental DNA or eDNA is a promising tool to detect species in environments 34 such as aquatic systems, where specimen capture can demand effort and time. Although eDNA 35 can be substantially informative, many factors may affect the quantity and quality of eDNA. Such 36 factors involve DNA degradation, DNA contamination, DNA collecting protocols, and PCR 37 inhibitors, etc. The purpose of this dissertation is to establish a protocol in a controlled tropical 38 environment: aquariums that contain the target fish Prochilodus argenteus. The water from the 39 aquarium was filtered and the DNA was extracted with the QIAamp DNA Stool Mini Kit and 40 Species-specific primers for P. argenteus were tested to amplify fragments of the D-loop region 41 of the mitochondrial DNA using two reactions: a direct or simple PCR and a Nested PCR with 42 internal primers, using products of the direct reaction as template. Other protocols involved 43 testing the effect of possible inhibitors from different water sources on the PCR, eliminating PCR 44 inhibitors through DNA purification protocols and testing DNA yield through filtration. Our results 45 suggest that the different types of water influence PCR results. The PCR using target DNA and 46 ultrapure water amplified from 1L of 15 ng/L of DNA to 0.00015 ng/L of DNA while the 47 reactions tested with water from the tropical aquariums amplified from 15 ng/L of DNA to 0.015 48 ng/L of DNA, whereas water from Pampulha lake amplified successfully only using 15 ng/L of 49 DNA. Protocols that included activated charcoal and Chelex 100 for DNA purification gave 50 inconsistent results and could not be replicated. Our results indicate that BSA is an efficient PCR 51 inhibitor blocker. We conclude that the water filtration protocol using GL filter allied to DNA 52 extraction through the QIAamp Stool Mini Kit was efficient to concentrate and eliminate inhibitors 53 that might cause under detection of eDNA.