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

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

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

10.21203/rs.3.rs-264679/v1 ◽

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.

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Effect of Extraction Systems on Clomazone Recovery from Aged Soil Samples

Journal of AOAC International ◽

10.1093/jaoac/78.6.1519 ◽

1995 ◽

Vol 78 (6) ◽

pp. 1519-1521 ◽

Cited By ~ 2

Author(s):

K Bruce Kirksey ◽

Thomas C Mueller

Keyword(s):

Liquid Chromatography ◽

Limit Of Detection ◽

Extraction Methods ◽

Reversed Phase ◽

Soil Samples ◽

Moist Soil ◽

Reversed Phase Liquid Chromatography ◽

Extraction Solvent ◽

Rotary Evaporator ◽

Phase Liquid

Abstract Seven extraction methods were examined to determine which one provided a simple, efficient clomazone extraction from aged soil. The method selected involved adding 80 mL acetonitrile to moist soil and shaking for 16 h. The extract was filtered, and an additional 80 mL acetonitrile was added, equilibrated for 1 h, and filtered. Filtrates were combined and concentrated with a rotary evaporator. Clomazone concentrations were then determined by reversed-phase liquid chromatography with UV detection at 220 nm. Recovery with this method was equal to a 16 h + 1 h extraction with methanol, but acetonitrile was selected as extraction solvent because of its ease of removal with a rotary evaporator. The recovery from fortified soil samples was >80%, and a conservative lower limit of detection was 40 ng/g soil.

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Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

Open Medicine ◽

10.2478/s11536-007-0069-4 ◽

2008 ◽

Vol 3 (2) ◽

pp. 157-162

Author(s):

Koray Ergunay ◽

Pinar Yurdakul ◽

Burcin Sener ◽

Ugur Ozcelik ◽

Erdem Karabulut ◽

...

Keyword(s):

Dna Extraction ◽

Burkholderia Cepacia ◽

Limit Of Detection ◽

Direct Detection ◽

Extraction Methods ◽

Rapid Identification ◽

Burkholderia Cepacia Complex ◽

Assay Sensitivity ◽

Spin Column ◽

Significant Difference

AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

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Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples

Molecular and Cellular Probes ◽

10.1016/j.mcp.2006.08.003 ◽

2007 ◽

Vol 21 (2) ◽

pp. 92-96 ◽

Cited By ~ 70

Author(s):

Chris A. Whitehouse ◽

Hannah E. Hottel

Keyword(s):

Dna Extraction ◽

Francisella Tularensis ◽

Soil Samples ◽

Spiked Soil

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Molecular characterization of Toxocara spp. eggs isolated from public parks and playgrounds in Shiraz, Iran

Journal of Helminthology ◽

10.1017/s0022149x18000354 ◽

2018 ◽

Vol 93 (3) ◽

pp. 306-312 ◽

Cited By ~ 3

Author(s):

M. Choobineh ◽

F. Mikaeili ◽

S.M. Sadjjadi ◽

S. Ebrahimi ◽

S. Iranmanesh

Keyword(s):

Toxocara Canis ◽

Human Infection ◽

Soil Samples ◽

Its Rdna ◽

Public Parks ◽

Toxocara Cati ◽

Nested Polymerase Chain Reaction ◽

Pcr Rflp ◽

Zinc Sulphate Solution ◽

Paratenic Hosts

AbstractHuman toxocariasis, a worldwide parasitic disease, is caused by the larval stage of intestinal nematodes of dogs and cats, namely Toxocara canis and Toxocara cati. Human infection occurs by the accidental ingestion of embryonated eggs present in the soil, vegetables or on other contaminated surfaces, as well as via consumption of uncooked paratenic hosts, such as bird meat and giblets. The objective of this study was to evaluate the contamination of soil in public parks and playgrounds in Shiraz using microscopy and molecular methods. A total of 150 soil samples were collected from public parks and playgrounds in various areas of Shiraz, southern Iran. The samples were treated with saturated zinc sulphate solution, and Toxocara spp. eggs were detected by microscopic observation followed by nested polymerase chain reaction (PCR). To differentiate T. canis and T. cati eggs from each other, PCR restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS)-rDNA region by SalI endonuclease enzyme was used. PCR-sequencing was performed to confirm the results of the PCR-RFLP method. Based on the flotation results of the 150 soil samples, six (4%) were found to be positive for Toxocara spp. eggs, whereas nested-PCR showed 24 samples to be positive (16%). Based on the PCR-RFLP method and the sequence of the ITS-rDNA region, a total of 23 out of 24 isolates were confirmed as T. cati and one out of 24 as T. canis. The results showed a higher number of soil samples to be positive for Toxocara by the molecular method than microscopy, and higher T. cati infection in soil samples, which could have an important role in human infection with toxocariasis in this region.

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Evaluation of Different Oocyst DNA Extraction Methods for Cryptosporidium spp. Research in Environmental Samples

Acta Parasitologica ◽

10.1007/s11686-020-00235-w ◽

2020 ◽

Vol 65 (4) ◽

pp. 995-998

Author(s):

Neliane Cristina Moreira ◽

Marlene Cabrine-Santos ◽

Márcia Benedita de Oliveira-Silva

Keyword(s):

Dna Extraction ◽

Environmental Samples ◽

Extraction Methods ◽

Cryptosporidium Spp ◽

Dna Extraction Methods

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Evaluation of DNA Extraction Methods and Bioinformatic Pipelines for Marine Nano- and Pico-Eukaryotic Plankton Analysis

Frontiers in Marine Science ◽

10.3389/fmars.2020.584253 ◽

2021 ◽

Vol 7 ◽

Author(s):

Marta Muñoz-Colmenero ◽

Ana Sánchez ◽

Begoña Correa ◽

Francisco G. Figueiras ◽

Jose L. Garrido ◽

...

Keyword(s):

Dna Extraction ◽

Environmental Sample ◽

Environmental Samples ◽

Extraction Methods ◽

Ion Torrent ◽

Taxonomic Assignment ◽

Dna Quality ◽

Eukaryotic Species ◽

Dna Extraction Methods ◽

Mock Communities

The smallest size fractions of plankton, nano- and pico-plankton, have been highlighted due to they accomplish key functions in marine ecosystems. However, the knowledge about some of them is scarce because they are difficult or impossible to be detected and identified with non-DNA-based methodologies. Here we have evaluated five DNA extraction protocols (MT1–MT5) and seven bioinformatic pipelines (P1–P7) to find the best protocol for detecting most of the eukaryotic species of nano- and pico-plankton present in an environmental sample using Ion Torrent technology. The protocol MT3 was the most reproducible methodology, showing less variation among samples, good DNA quality and sufficient quantity to amplify and sequence the eukaryote species, offering the best results after sequencing. For bioinformatic analyses, P1 and P7 resulted in the highest percentage of detection for the difficult-to-detect species in mock communities. However, only P1 avoided the confusion with other closed species during the taxonomic assignment. The final protocols, MT3-P1 (free) and MT3-P7 (private), showed good and consistent results when they were applied to an environmental sample, being a valuable tool to study the eukaryotes present in environmental samples of nano- and pico-plankton, even for the genera that are difficult to be detected by other techniques.

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DNA extraction

10.1093/oso/9780198767220.003.0005 ◽

2018 ◽

Author(s):

Pierre Taberlet ◽

Aurélie Bonin ◽

Lucie Zinger ◽

Eric Coissac

Keyword(s):

Dna Extraction ◽

Phosphate Buffer ◽

Environmental Samples ◽

Soil Samples ◽

Mobile Laboratory ◽

Extraction Protocol ◽

Soil Sediment ◽

Total Dna ◽

Practical Constraints ◽

Detailed Protocol

Chapter 5 “DNA extraction” focuses on the particularities and practical constraints associated with the isolation of eDNA from environmental samples. The extraction protocol is indeed crucial in eDNA studies, as it will determine whether extracellular, intracellular, or total DNA is targeted. Chapter 5 describes the main advantages and limitations of the most popular extraction kits aimed at obtaining DNA from soil, sediment, litter, feces, or water. It provides a detailed protocol for DNA extraction from soil samples using a saturated phosphate buffer. This protocol has been optimized for an easy implementation in the field using a mobile laboratory, so the material and consumables necessary are also listed.

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