Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capilla...

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Autores principales:	Bettina Thalinger, Yannick Pütz, Michael Traugott
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4e50a0166b974ebe8a2927610bd39228
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spelling	oai:doaj.org-article:4e50a0166b974ebe8a2927610bd392282021-12-02T20:04:54ZEndpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.1932-620310.1371/journal.pone.0254356https://doaj.org/article/4e50a0166b974ebe8a2927610bd392282021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0254356https://doaj.org/toc/1932-6203The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.Bettina ThalingerYannick PützMichael TraugottPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 7, p e0254356 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Bettina Thalinger Yannick Pütz Michael Traugott Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.
description	The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.
format	article
author	Bettina Thalinger Yannick Pütz Michael Traugott
author_facet	Bettina Thalinger Yannick Pütz Michael Traugott
author_sort	Bettina Thalinger
title	Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.
title_short	Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.
title_full	Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.
title_fullStr	Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.
title_full_unstemmed	Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.
title_sort	endpoint pcr coupled with capillary electrophoresis (celpcr) provides sensitive and quantitative measures of environmental dna in singleplex and multiplex reactions.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/4e50a0166b974ebe8a2927610bd39228
work_keys_str_mv	AT bettinathalinger endpointpcrcoupledwithcapillaryelectrophoresiscelpcrprovidessensitiveandquantitativemeasuresofenvironmentaldnainsingleplexandmultiplexreactions AT yannickputz endpointpcrcoupledwithcapillaryelectrophoresiscelpcrprovidessensitiveandquantitativemeasuresofenvironmentaldnainsingleplexandmultiplexreactions AT michaeltraugott endpointpcrcoupledwithcapillaryelectrophoresiscelpcrprovidessensitiveandquantitativemeasuresofenvironmentaldnainsingleplexandmultiplexreactions
_version_	1718375539924795392

Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions.

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