Optimized DNA-based Identification of Toxocara spp. Egg in Difficult Matrices: A Case for Specific and Sensitive Identification of Geohelminth Eggs

2021 ◽  
Author(s):  
Wojciech Jarosz ◽  
Jean-François Durant ◽  
Leonid Mwana wa bene Irenge ◽  
Renata Fogt-Wyrwas ◽  
Hanna Mizgajska-Wiktor ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Limit Of Detection ◽  
Toxocara Canis ◽  
Toxocara Cati ◽  
Flow Process ◽  
Extraction And Purification ◽  
Causative Agents ◽  
Environment Control ◽  
Set Up

Abstract 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.

scholarly journals Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Wojciech Jarosz ◽  
Jean-Francois Durant ◽  
Leonid Mwana Wa Bene Irenge ◽  
Renata Fogt-Wyrwas ◽  
Hanna Mizgajska-Wiktor ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Limit Of Detection ◽  
Toxocara Canis ◽  
Extraction Methods ◽  
Soil Samples ◽  
Toxocara Cati ◽  
Flow Process ◽  
Spiked Soil ◽  
Set Up

Abstract 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 104 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

Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati

Parasitology ◽  
2021 ◽  
pp. 1-8
Author(s):  
Héctor Gabriel Avila ◽  
Marikena Guadalupe Risso ◽  
Paula Ruybal ◽  
Silvia Analía Repetto ◽  
Marcos Javier Butti ◽  
...  
Keyword(s):  
Low Cost ◽  
Lamp Assay ◽  
Toxocara Canis ◽  
Toxocara Cati

Abstract

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension)

The Analyst ◽  
2012 ◽  
Vol 137 (17) ◽  
pp. 4023 ◽  
Author(s):  
Lindsay N. Strotman ◽  
Guangyun Lin ◽  
Scott M. Berry ◽  
Eric A. Johnson ◽  
David J. Beebe
Keyword(s):  
Surface Tension ◽  
Dna Extraction ◽  
Extraction And Purification ◽  
Food Matrices

scholarly journals Detection of Bacteria in Environmental Samples by Direct PCR Without DNA Extraction

BioTechniques ◽  
2001 ◽  
Vol 31 (3) ◽  
pp. 598-607 ◽  
Author(s):  
Kimberly A. Fode-Vaughan ◽  
Charles F. Wimpee ◽  
Charles C. Remsen ◽  
Mary Lynne Perille Collins
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Direct Pcr

Distribution of Toxocara infection in the environment and in definitive and paratenic hosts in Estonia

2006 ◽  
Vol 54 (3) ◽  
pp. 399-406 ◽  
Author(s):  
Heli Talvik ◽  
Epp Moks ◽  
Erika Mägi ◽  
T. Järvis ◽  
Illa Miller
Keyword(s):  
Rattus Norvegicus ◽  
Risk Group ◽  
Toxocara Canis ◽  
Toxocara Cati ◽  
Low Level ◽  
Larval Infection ◽  
Small Intestines ◽  
Paratenic Hosts ◽  
The Town ◽  
Home Territory

The aim of the study was to elucidate the distribution and possible transmission routes of Toxocara spp. infection in Estonia. Out of 454 faecal and sand samples collected from park lawns and sandpits in the town of Tartu, 19 were Toxocara positive (4.2%). Out of the 45 sandpit samples 17.8% were Toxocara positive. Cat faeces was found in 21 sandpit samples. Parasitological necropsies were performed on 41 euthanised stray dogs and 27 cats in the Tallinn Dog Home. Additionally, 13 wild free-roaming brown rats (Rattus norvegicus) were captured from the Tallinn Dog Home territory, necropsied and studied for the presence of Toxocara larvae. Toxocara canis adults were found in 14.6% of the dogs and Toxocara cati (syn. mystax) adults in the small intestines of 48.2% of the cats examined. Larval infection was detected in the kidney and liver in 5 dogs (12.2%). Our study demonstrated only low-level larval Toxocara infections in adult dogs. Toxocara larvae were not found in cats and brown rats. According to the results of this study, cats more often carry Toxocara infection than dogs. Under our conditions, stray and free-roaming cats are the main contaminators of the environment with Toxocara eggs. Children playing in sandpits are the main risk group for larval toxocarosis.

A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure

2003 ◽  
Vol 54 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Teegan Trochimchuk ◽  
John Fotheringham ◽  
Edward Topp ◽  
Heidi Schraft ◽  
Kam Tin Leung
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Cattle Manure ◽  
Escherichia Coli O157 ◽  
Extraction And Purification ◽  
Purification Methods

Extracellular DNA in environmental samples: Occurrence, extraction, quantification, and impact on microbial biodiversity assessment

2021 ◽  
Author(s):  
Sakcham Bairoliya ◽  
Jonas Koh Zhi Xiang ◽  
Bin Cao
Keyword(s):  
Dna Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Environmental Dna ◽  
Extracellular Dna ◽  
Biodiversity Assessment ◽  
Microbial Biodiversity ◽  
Technical Details ◽  
The Impact

Environmental DNA, i.e., DNA directly extracted from environmental samples, has been applied to understand microbial communities in the environments and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggest several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by: (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods including qPCR and fluorescent DNA binding dyes; (iii) carefully interpretating effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses.

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