Cargando…
Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples
BACKGROUND: Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potential...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390219/ https://www.ncbi.nlm.nih.gov/pubmed/34446077 http://dx.doi.org/10.1186/s13071-021-04904-1 |
_version_ | 1783743046051954688 |
---|---|
author | Jarosz, Wojciech Durant, Jean-Francois Irenge, Leonid Mwana Wa Bene Fogt-Wyrwas, Renata Mizgajska-Wiktor, Hanna Gala, Jean-Luc |
author_facet | Jarosz, Wojciech Durant, Jean-Francois Irenge, Leonid Mwana Wa Bene Fogt-Wyrwas, Renata Mizgajska-Wiktor, Hanna Gala, Jean-Luc |
author_sort | Jarosz, Wojciech |
collection | PubMed |
description | BACKGROUND: Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. METHODS: To select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy(®) PowerMax(®) Soil Kit) with an optional dilution and/or clean-up (Agencourt(®) AMPure(®)) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 10(4) eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs. RESULTS: The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy(®) PowerMax(®) Soil Kit, and completed with an additional DNA clean-up step with AMPure(®) beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T. canis eggs in 10-g sand and 10-g soil samples, respectively. CONCLUSIONS: The pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13071-021-04904-1. |
format | Online Article Text |
id | pubmed-8390219 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-83902192021-08-27 Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples Jarosz, Wojciech Durant, Jean-Francois Irenge, Leonid Mwana Wa Bene Fogt-Wyrwas, Renata Mizgajska-Wiktor, Hanna Gala, Jean-Luc Parasit Vectors Research BACKGROUND: Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. METHODS: To select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy(®) PowerMax(®) Soil Kit) with an optional dilution and/or clean-up (Agencourt(®) AMPure(®)) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 10(4) eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs. RESULTS: The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy(®) PowerMax(®) Soil Kit, and completed with an additional DNA clean-up step with AMPure(®) beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T. canis eggs in 10-g sand and 10-g soil samples, respectively. CONCLUSIONS: The pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13071-021-04904-1. BioMed Central 2021-08-26 /pmc/articles/PMC8390219/ /pubmed/34446077 http://dx.doi.org/10.1186/s13071-021-04904-1 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Jarosz, Wojciech Durant, Jean-Francois Irenge, Leonid Mwana Wa Bene Fogt-Wyrwas, Renata Mizgajska-Wiktor, Hanna Gala, Jean-Luc Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples |
title | Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples |
title_full | Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples |
title_fullStr | Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples |
title_full_unstemmed | Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples |
title_short | Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples |
title_sort | optimized dna-based identification of toxocara spp. eggs in soil and sand samples |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390219/ https://www.ncbi.nlm.nih.gov/pubmed/34446077 http://dx.doi.org/10.1186/s13071-021-04904-1 |
work_keys_str_mv | AT jaroszwojciech optimizeddnabasedidentificationoftoxocarasppeggsinsoilandsandsamples AT durantjeanfrancois optimizeddnabasedidentificationoftoxocarasppeggsinsoilandsandsamples AT irengeleonidmwanawabene optimizeddnabasedidentificationoftoxocarasppeggsinsoilandsandsamples AT fogtwyrwasrenata optimizeddnabasedidentificationoftoxocarasppeggsinsoilandsandsamples AT mizgajskawiktorhanna optimizeddnabasedidentificationoftoxocarasppeggsinsoilandsandsamples AT galajeanluc optimizeddnabasedidentificationoftoxocarasppeggsinsoilandsandsamples |