Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool.
BACKGROUND:A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the...
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ftdoajarticles:oai:doaj.org/article:c69a4a4354714974a628557b87ddd08f 2023-05-15T15:14:11+02:00 Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. Mio Ayana Piet Cools Zeleke Mekonnen Abdissa Biruksew Daniel Dana Nour Rashwan Roger Prichard Johnny Vlaminck Jaco J Verweij Bruno Levecke 2019-10-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0007778 https://doaj.org/article/c69a4a4354714974a628557b87ddd08f EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0007778 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0007778 https://doaj.org/article/c69a4a4354714974a628557b87ddd08f PLoS Neglected Tropical Diseases, Vol 13, Iss 10, p e0007778 (2019) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2019 ftdoajarticles https://doi.org/10.1371/journal.pntd.0007778 2022-12-31T13:13:07Z BACKGROUND:A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. METHODOLOGY AND PRINCIPAL FINDINGS:In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNAlater and were stored at 4°C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. CONCLUSIONS:The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 13 10 e0007778 |
institution |
Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Mio Ayana Piet Cools Zeleke Mekonnen Abdissa Biruksew Daniel Dana Nour Rashwan Roger Prichard Johnny Vlaminck Jaco J Verweij Bruno Levecke Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
BACKGROUND:A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. METHODOLOGY AND PRINCIPAL FINDINGS:In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNAlater and were stored at 4°C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. CONCLUSIONS:The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs. |
format |
Article in Journal/Newspaper |
author |
Mio Ayana Piet Cools Zeleke Mekonnen Abdissa Biruksew Daniel Dana Nour Rashwan Roger Prichard Johnny Vlaminck Jaco J Verweij Bruno Levecke |
author_facet |
Mio Ayana Piet Cools Zeleke Mekonnen Abdissa Biruksew Daniel Dana Nour Rashwan Roger Prichard Johnny Vlaminck Jaco J Verweij Bruno Levecke |
author_sort |
Mio Ayana |
title |
Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. |
title_short |
Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. |
title_full |
Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. |
title_fullStr |
Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. |
title_full_unstemmed |
Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. |
title_sort |
comparison of four dna extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2019 |
url |
https://doi.org/10.1371/journal.pntd.0007778 https://doaj.org/article/c69a4a4354714974a628557b87ddd08f |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 13, Iss 10, p e0007778 (2019) |
op_relation |
https://doi.org/10.1371/journal.pntd.0007778 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0007778 https://doaj.org/article/c69a4a4354714974a628557b87ddd08f |
op_doi |
https://doi.org/10.1371/journal.pntd.0007778 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
13 |
container_issue |
10 |
container_start_page |
e0007778 |
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1766344671647236096 |