A simple and selective electrochemical magneto-assay for sea lice eDNA detection developed with a Quality by Design approach.
Sci Total Environ
; 791: 148111, 2021 Oct 15.
Article
en En
| MEDLINE
| ID: mdl-34119793
ABSTRACT
Environmental DNA (eDNA) is a novel, non-invasive sampling procedure that allows the obtaining of genetic material directly from environmental samples without any evidence of biological sources. The eDNA methodology can greatly benefit from coupling it to reliable, portable and cost-effective tools able to perform decentralized measurements directly at the site of need and in resource-limited settings. Herein, we report a simple method for the selective analysis of eDNA using a magneto-assay with electrochemical detection. The proposed method involves the polymerase chain-reaction (PCR) amplification of mitochondrial eDNA of parasitic Salmon lice (Lepeophtheirus salmonis), extracted from seawater samples. The eDNA sequence was targeted via sandwich hybridization onto magnetic beads and enzymatic labeling was performed to obtain an electroactive product measured by differential pulse voltammetry. Quality by Design (QbD), a recent concept of science- and risk-oriented quality paradigm, was used for the optimization of the different parameters of the assay. Response surface methodology and Monte Carlo simulations were performed to define the method operable design region. The optimized electrochemical magneto-assay attained a limit of detection of 2.9 amol µL-1 of the short synthetic sea louse DNA analogue (43 bp). In addition, robustness testing using a further experimental design approach was performed for monitoring eDNA amplicons. Seawater samples spiked with individuals of free-swimming L. salmonis copepodite stages and seawater collected from tanks with sea lice-infested fish were analyzed.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Salmo salar
/
Copépodos
/
Enfermedades de los Peces
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Sci Total Environ
Año:
2021
Tipo del documento:
Article
País de afiliación:
Italia