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Comparative assessment of DNA extraction procedures for Ascaris spp. eggs

Published online by Cambridge University Press:  28 August 2019

I.D. Amoah Open the ORCID record for I.D. Amoah [Opens in a new window] ,
G. Singh ,
K. Troell ,
P. Reddy ,
T.A. Stenström  and
F. Bux
I.D. Amoah*
Affiliation:
Institute for Water and Wastewater Technology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa
G. Singh
Affiliation:
Institute for Water and Wastewater Technology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa
K. Troell
Affiliation:
Department of Microbiology, National Veterinary Institute, SE-751 89, Uppsala, Sweden
P. Reddy
Affiliation:
Department of Community Health Studies, Faculty of Health Sciences, Durban University of Technology, PO Box 1334, Durban 4000, South Africa
T.A. Stenström
Affiliation:
Institute for Water and Wastewater Technology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa
F. Bux
Affiliation:
Institute for Water and Wastewater Technology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa
*
Author for correspondence: I.D. Amoah, E-mail: amoahkid@gmail.com
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Abstract

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

Keywords

Soil-transmitted helminthsAscaris suumqPCRNanoDropQubitPCR inhibitorsDNA extraction kits
Type
Research Paper
Information
Journal of Helminthology , Volume 94 , 2020 , e78
Copyright
Copyright © Cambridge University Press 2019 

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References

Abreu-Acosta, N and Vera, L (2011) Occurrence and removal of parasites, enteric bacteria and faecal contamination indicators in wastewater natural reclamation systems in Tenerife-Canary Islands, Spain. Ecological Engineering 37, 496–503.Google Scholar
Ai, L, Dong, SJ, Zhang, WY, Elsheikha, HM, Mahmmod, YS and Lin, RQ (2010) Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys. Annals of Tropical Medicine and Parasitology 104, 65–72.Google Scholar
Amoah, ID, Singh, G, Stenström, TA and Reddy, P (2017) Detection and quantification of soil-transmitted helminths in environmental samples: a review of current state-of-the-art and future perspectives. Acta Tropica 169, 187–201.Google Scholar
Ariefdjohan, MW, Savaiano, DA and Nakatsu, CH (2010) Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. Journal of Nutrition 9, 23–31.Google Scholar
Basuni, M, Muhi, J, Othman, N, et al. (2011) A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths. American Journal of Tropical Medicine and Hygiene 84, 338–343.Google Scholar
Boesenberg-Smith, KA, Pessarakli, MM and Wolk, DM (2012) Assessment of DNA yield and purity: an overlooked detail of PCR troubleshooting. Clinical Microbiology Newsletter 34, 1–5.Google Scholar
Collender, PA, Kirby, AE, Addiss, DG, Freeman, MC and Remais, JV (2015) Methods for quantification of soil-transmitted helminths in environmental media: current techniques and recent advances. Trends in Parasitology 31, 623–639.Google Scholar
Dietrich, D, Uhl, B, Sailer, V, Holmes, EE and Jung, M (2013) Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition. PLoS One 8, e77771.Google Scholar
Forslund, A, Ensink, JHJ, Battilani, A, et al. (2010) Faecal contamination and hygiene aspect associated with the use of treated wastewater and canal water for irrigation of potatoes (Solanum tuberosum). Agricultural Water Management 98, 440–450.Google Scholar
Fortin, N, Beaumier, D, Lee, K and Greer, CW (2004) Soil washing improves the recovery of total community DNA from polluted and high organic content sediments. Journal of Microbiology Methods 56, 181–191.Google Scholar
Gilbert, MT, Haselkorn, T, Bunce, M, Sanchez, JJ and Lucas, SB (2007) The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PLoS One 2, e537.Google Scholar
Gordon, CA, McManus, DP, Acosta, LP, Olveda, RM, Williams, GM, Ross, AG, Gray, DJ and Gobert, GN (2015) Multiplex real-time PCR monitoring of intestinal helminths in humans reveals widespread polyparasitism in Northern Samar, the Philippines. International Journal of Parasitology 45, 477–483.Google Scholar
Grimes, JET, Tadesse, G, Mekete, K, et al. (2016) School water, sanitation, and hygiene, Soil-Transmitted Helminths, and Schistosomes: national mapping in Ethiopia. PLoS Neglected Tropical Diseases 10(3), e0004515.Google Scholar
Gyawali, P, Ahmed, W, Sidhu, JP, Jagals, P and Toze, S (2017) Quantification of hookworm ova from wastewater matrices using quantitative PCR. Journal of Environmental Science 57, 231–237.Google Scholar
Hall, TA, Zovanyi, MA, Christensen, DR, Koehler, JW and Devins, MT (2013) Evaluation of Inhibitor-Resistant Real-Time PCR methods for diagnostics in clinical and environmental samples. PLoS One 8(9), e73845.Google Scholar
Josefsen, MH, Andersen, SC, Christensen, J and Hoorfar, J (2015) Microbial food safety: potential of DNA extraction methods for use in diagnostic metagenomics. Journal of Microbiology Methods 114, 30–34.Google Scholar
Lakay, FM, Botha, A and Prior, BA (2006) Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils. Journal of Applied Microbiology 102, 265–273.Google Scholar
Luo, HQ, Zhang, H, Li, K, et al. (2017) Molecular characterization of ascaris from Tibetan pigs by three mitochondrial markers of nad1, cox1 and cox2. Tropical Biomedicine 34, 576–582.Google Scholar
Miller, DN, Bryant, JE, Madsen, EL and Ghiorse, WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Applied and Environmental Microbiology 65, 4715–4724.Google Scholar
Naidoo, D, Archer, C, Louton, B and Rodda, N (2016) Testing household disinfectants for the inactivation of helminth eggs on surfaces and in spills during pit latrine emptying. Water SA 42, 560–570.Google Scholar
Olson, ND and Morrow, JB (2012) DNA extract characterization process for microbial detection methods development and validation. BMC Research Notes 5, 668–682.Google Scholar
Pecson, BM, Barrios, JA, Johnson, DR and Nelson, KL (2006) A realtime PCR method for quantifying viable Ascaris eggs using the first internally transcribed spacer region of ribosomal DNA. Applied and Environmental Microbiology 72, 7864–7872.Google Scholar
Quilès, F, Balandier, JY and Capizzi-Banas, S (2006) In situ characterisation of a microorganism surface by Raman microspectroscopy: the shell of Ascaris eggs. Analytical and Bioanalytical Chemistry 386, 249–255.Google Scholar
Rostami, A, Ebrahimi, M, Mehravar, S, Omrani, VF, Fallahi, S and Behniafar, H (2016) Contamination of commonly consumed raw vegetables with soil transmitted helminth eggs in Mazandaran province, Northern Iran. International Journal of Food Microbiology 225, 54–58.Google Scholar
Salonen, A, Nikkilä, J, Jalanka-Tuovinen, J, Immonen, O, Rajilić-Stojanović, M, Kekkonen, RA, Palva, A and de Vos, WM (2010) Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. Journal of Microbiology Methods 81, 127–134.Google Scholar
Scaglia, B, D'Imporzano, G, Garuti, G, Negri, M and Adani, F (2014) Sanitation ability of anaerobic digestion performed at different temperature on sewage sludge. Science of the Total Environment 466–467, 888–897.Google Scholar
Sørensen, SJ, Müller, AK, Hansen, LH, Rasmussen, LD, Lipthay, JR and Barkay, T (2002) Molecular methods for assessing and manipulating the diversity of microbiological populations and processes. pp. 289–294 in Burns, RG, Dick, RP (Eds) Enzymes in the environment. New York, Marcel Dekker.Google Scholar
Tebbe, CC and Vahjen, W (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant-DNA from bacteria and a yeast. Applied and Environmental Microbiology 59, 2657–2665.Google Scholar
Thermo Scientific (2010) Thermo scientific NanoDrop spectrophotometers: nucleic acid. Product description. MA, USA, Thermo Fisher Scientific Inc.Google Scholar
Valero, MA, Perez-Crespo, I, Periago, MV, Khoubbane, M and Mas-Coma, S (2009) Fluke egg characteristics for the diagnosis of human and animal fascioliasis by Fasciola hepatica and F gigantica. Acta Tropica 111, 150–159.Google Scholar
Verweij, JJ, Brienen, EA, Ziem, J, Yelifari, L, Polderman, AM and Van Lieshout, L (2007) Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. American Journal of Tropical Medicine and Hygiene 77, 685–690.Google Scholar
Verweij, JJ, Canales, M, Polman, K, Ziem, J, Brienen, EA, Polderman, AM and van Lieshout, L (2009) Molecular diagnosis of Strongyloides stercoralis in fecal samples using real-time PCR. Transactions of the Royal Society of Tropical Medicine and Hygiene 103, 342–346.Google Scholar
Wiria, AE, Prasetyani, MA, Hamid, F, et al. (2010) Does treatment of intestinal helminth infections influence malaria? Background and methodology of a longitudinal study of clinical, parasitological and immunological parameters in Nangapanda, Flores, Indonesia (ImmunoSPIN Study). BMC Infectious Diseases 10, 1–12.Google Scholar
Yen-Phi, VT, Rechenburg, A, Vinnerås, B, Clemens, J and Kistemann, T (2010) Pathogens in septage in Vietnam. Science of the Total Environment 408, 2050–2053.Google Scholar
Zhou, JZ and Thompson, DK (2002) Challenges in applying microarrays to environmental studies. Current Opinions in Biotechnology 13, 204–207.Google Scholar