| Autor: |
Hanna Mizgajska-Wiktor, W. Jarosz, Leonid M. Irenge, Jean-Luc Gala, Jean-François Durant, Renata Fogt-Wyrwas |
| Rok vydání: |
2021 |
| Předmět: |
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| DOI: |
10.21203/rs.3.rs-264679/v1 |
| Popis: |
BackgroundToxocara canis (T. canis) and Toxocara cati (T. cati) are worldwide-distributed roundworms of canids and felids and causative agents of human toxocarosis, via ingestion of Toxocara eggs disseminated in the environment. Control of Toxocara infections is constrained by the lack of sensitive methods for screening of animal feces and environmental samples potentially contaminated by Toxocara eggs. We previously developed a quantitative duplex real-time PCR (qPCR) for sensitive and specific detection of T. canis and T. cati. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs present in environmental samples was set up. For this purpose, the influence of different methods for eggs lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples.MethodsSoil and sand (10g) samples were spiked with egg suspensions of T. canis. DNA was extracted from Toxocara eggs, using different DNA extraction kits (FastDNA™ SPIN Kit for Soil and DNeasy® PowerMax® Soil Kit), and an additional clean-up step (Agencourt® AMPure®). The efficiency of the above-developed process was compared with the conventional flotation-centrifugation and observation of Toxocara eggs under light microscopy.ResultsThe most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, DNA extraction with the DNeasy® PowerMax® Soil Kit and an additional DNA clean-up step with AMPure® beads. with a limit of detection of 6 eggs of T. canis spiked in 10 g of soil with a probability of 97%.ConclusionThe pre-analytical flow process developed here combined with qPCR represents an improved method for the surveillance of Toxocara contamination in the environment. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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