Cargando…

Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals

Although field-sampling procedures to capture gDNA from individual schistosome larval stages directly from their natural hosts exist, they do pose some technical and logistical challenges hampering certain epidemiological studies. The aim of this study was to develop, refine and evaluate an alternat...

Descripción completa

Detalles Bibliográficos
Autor principal: Webster, Bonnie L
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770516/
https://www.ncbi.nlm.nih.gov/pubmed/19852777
http://dx.doi.org/10.1186/1756-3305-2-50
_version_ 1782173673314058240
author Webster, Bonnie L
author_facet Webster, Bonnie L
author_sort Webster, Bonnie L
collection PubMed
description Although field-sampling procedures to capture gDNA from individual schistosome larval stages directly from their natural hosts exist, they do pose some technical and logistical challenges hampering certain epidemiological studies. The aim of this study was to develop, refine and evaluate an alternative methodology, which enables better preservation of large numbers of individual schistosome larval stages and eggs collected in low resource endemic areas, to provide PCR-quality DNA for multi-locus genetic analysis. The techniques reported here present simple and effective short-term field and long-term laboratory preservation and storage systems for individually sampled schistosome eggs and larval stages using a commercially available aqueous stabilisation reagent, RNAlater(® )eliminating the need for more cumbersome resources such as refrigerators, heaters and centrifuge equipment for immediate specimen processing. Adaptations to a general gDNA extraction method are described, that enables the acquisition of a gDNA extract (~50 μl), facilitating multiple molecular analyses of each sampled schistosome. The methodology provided PCR-quality mitochondrial and nuclear DNA from laboratory cercariae, miracidia and eggs that had been stored at up to 37°C for 2 weeks and at 4°C for 6 months and also from field collected samples. This present protocol provides significant epidemiological, ethical and practical advantages over existing sampling methods and has the potential to be transferred to studies on other organisms, especially where specimens are unable to be seen by the naked eye, are difficult to handle and need to be obtained from a field environment.
format Text
id pubmed-2770516
institution National Center for Biotechnology Information
language English
publishDate 2009
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-27705162009-10-30 Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals Webster, Bonnie L Parasit Vectors Short Report Although field-sampling procedures to capture gDNA from individual schistosome larval stages directly from their natural hosts exist, they do pose some technical and logistical challenges hampering certain epidemiological studies. The aim of this study was to develop, refine and evaluate an alternative methodology, which enables better preservation of large numbers of individual schistosome larval stages and eggs collected in low resource endemic areas, to provide PCR-quality DNA for multi-locus genetic analysis. The techniques reported here present simple and effective short-term field and long-term laboratory preservation and storage systems for individually sampled schistosome eggs and larval stages using a commercially available aqueous stabilisation reagent, RNAlater(® )eliminating the need for more cumbersome resources such as refrigerators, heaters and centrifuge equipment for immediate specimen processing. Adaptations to a general gDNA extraction method are described, that enables the acquisition of a gDNA extract (~50 μl), facilitating multiple molecular analyses of each sampled schistosome. The methodology provided PCR-quality mitochondrial and nuclear DNA from laboratory cercariae, miracidia and eggs that had been stored at up to 37°C for 2 weeks and at 4°C for 6 months and also from field collected samples. This present protocol provides significant epidemiological, ethical and practical advantages over existing sampling methods and has the potential to be transferred to studies on other organisms, especially where specimens are unable to be seen by the naked eye, are difficult to handle and need to be obtained from a field environment. BioMed Central 2009-10-23 /pmc/articles/PMC2770516/ /pubmed/19852777 http://dx.doi.org/10.1186/1756-3305-2-50 Text en Copyright © 2009 Webster; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Short Report
Webster, Bonnie L
Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals
title Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals
title_full Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals
title_fullStr Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals
title_full_unstemmed Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals
title_short Isolation and preservation of schistosome eggs and larvae in RNAlater(® )facilitates genetic profiling of individuals
title_sort isolation and preservation of schistosome eggs and larvae in rnalater(® )facilitates genetic profiling of individuals
topic Short Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770516/
https://www.ncbi.nlm.nih.gov/pubmed/19852777
http://dx.doi.org/10.1186/1756-3305-2-50
work_keys_str_mv AT websterbonniel isolationandpreservationofschistosomeeggsandlarvaeinrnalaterfacilitatesgeneticprofilingofindividuals