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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/138576
Ver los metadatos del registro completo
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CONICET Digital (CONICET) |
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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) |
collection |
CONICET Digital (CONICET) |
instname_str |
Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.name.fl_str_mv |
CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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1844613527541645312 |
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13.070432 |