Variably improved microbial source tracking with digital droplet PCR

Autores
Nshimyimana, Jean Pierre; Cruz, Mercedes Cecilia; Wuertz, Stefan; Thompson, Janelle R.
Año de publicación
2019
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85–1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74–100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.
Fil: Nshimyimana, Jean Pierre. Michigan State University; Estados Unidos. Massachusetts Institute of Technology; Estados Unidos. Nanyang Technological University; Singapur
Fil: Cruz, Mercedes Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; Argentina. Nanyang Technological University; Singapur
Fil: Wuertz, Stefan. Nanyang Technological University; Singapur
Fil: Thompson, Janelle R.. Massachusetts Institute of Technology; Estados Unidos
Materia
MICROBIAL SOURCE TRACKING
DIGITAL DROPLET PCR
QUANTITATIVE PCR
GENETIC MARKERS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/138576

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network_name_str CONICET Digital (CONICET)
spelling Variably improved microbial source tracking with digital droplet PCRNshimyimana, Jean PierreCruz, Mercedes CeciliaWuertz, StefanThompson, Janelle R.MICROBIAL SOURCE TRACKINGDIGITAL DROPLET PCRQUANTITATIVE PCRGENETIC MARKERShttps://purl.org/becyt/ford/2.8https://purl.org/becyt/ford/2This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85–1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74–100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.Fil: Nshimyimana, Jean Pierre. Michigan State University; Estados Unidos. Massachusetts Institute of Technology; Estados Unidos. Nanyang Technological University; SingapurFil: Cruz, Mercedes Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; Argentina. Nanyang Technological University; SingapurFil: Wuertz, Stefan. Nanyang Technological University; SingapurFil: Thompson, Janelle R.. Massachusetts Institute of Technology; Estados UnidosPergamon-Elsevier Science Ltd2019-08-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/138576Nshimyimana, Jean Pierre; Cruz, Mercedes Cecilia; Wuertz, Stefan; Thompson, Janelle R.; Variably improved microbial source tracking with digital droplet PCR; Pergamon-Elsevier Science Ltd; Water Research; 159; 1-8-2019; 192-2020043-1354CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S0043135419303732info:eu-repo/semantics/altIdentifier/doi/10.1016/j.watres.2019.04.056info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:49:18Zoai:ri.conicet.gov.ar:11336/138576instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:49:18.908CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Variably improved microbial source tracking with digital droplet PCR
title Variably improved microbial source tracking with digital droplet PCR
spellingShingle Variably improved microbial source tracking with digital droplet PCR
Nshimyimana, Jean Pierre
MICROBIAL SOURCE TRACKING
DIGITAL DROPLET PCR
QUANTITATIVE PCR
GENETIC MARKERS
title_short Variably improved microbial source tracking with digital droplet PCR
title_full Variably improved microbial source tracking with digital droplet PCR
title_fullStr Variably improved microbial source tracking with digital droplet PCR
title_full_unstemmed Variably improved microbial source tracking with digital droplet PCR
title_sort Variably improved microbial source tracking with digital droplet PCR
dc.creator.none.fl_str_mv Nshimyimana, Jean Pierre
Cruz, Mercedes Cecilia
Wuertz, Stefan
Thompson, Janelle R.
author Nshimyimana, Jean Pierre
author_facet Nshimyimana, Jean Pierre
Cruz, Mercedes Cecilia
Wuertz, Stefan
Thompson, Janelle R.
author_role author
author2 Cruz, Mercedes Cecilia
Wuertz, Stefan
Thompson, Janelle R.
author2_role author
author
author
dc.subject.none.fl_str_mv MICROBIAL SOURCE TRACKING
DIGITAL DROPLET PCR
QUANTITATIVE PCR
GENETIC MARKERS
topic MICROBIAL SOURCE TRACKING
DIGITAL DROPLET PCR
QUANTITATIVE PCR
GENETIC MARKERS
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.8
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85–1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74–100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.
Fil: Nshimyimana, Jean Pierre. Michigan State University; Estados Unidos. Massachusetts Institute of Technology; Estados Unidos. Nanyang Technological University; Singapur
Fil: Cruz, Mercedes Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones para la Industria Química. Universidad Nacional de Salta. Facultad de Ingeniería. Instituto de Investigaciones para la Industria Química; Argentina. Nanyang Technological University; Singapur
Fil: Wuertz, Stefan. Nanyang Technological University; Singapur
Fil: Thompson, Janelle R.. Massachusetts Institute of Technology; Estados Unidos
description This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85–1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74–100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ± 2.8 for B. theta and 18.7 ± 10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.
publishDate 2019
dc.date.none.fl_str_mv 2019-08-01
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/138576
Nshimyimana, Jean Pierre; Cruz, Mercedes Cecilia; Wuertz, Stefan; Thompson, Janelle R.; Variably improved microbial source tracking with digital droplet PCR; Pergamon-Elsevier Science Ltd; Water Research; 159; 1-8-2019; 192-202
0043-1354
CONICET Digital
CONICET
url http://hdl.handle.net/11336/138576
identifier_str_mv Nshimyimana, Jean Pierre; Cruz, Mercedes Cecilia; Wuertz, Stefan; Thompson, Janelle R.; Variably improved microbial source tracking with digital droplet PCR; Pergamon-Elsevier Science Ltd; Water Research; 159; 1-8-2019; 192-202
0043-1354
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S0043135419303732
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.watres.2019.04.056
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Pergamon-Elsevier Science Ltd
publisher.none.fl_str_mv Pergamon-Elsevier Science Ltd
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
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instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
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