Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR

Autores
Heckel, Sofía Belén; Pacini, Antonella; Paredes, Franco; Petreli, Maria Victoria; Perez, Marilina; Adriani, Natalia; Ibarra, Guadalupe; Menzella, Hugo Gabriel; Colaneri, Alejandro Cesar; Sesma, Juliana
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.
Fil: Heckel, Sofía Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina
Fil: Pacini, Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina
Fil: Paredes, Franco. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; Argentina
Fil: Petreli, Maria Victoria. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; Argentina
Fil: Perez, Marilina. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina
Fil: Adriani, Natalia. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina
Fil: Ibarra, Guadalupe. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; Argentina
Fil: Menzella, Hugo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina
Fil: Colaneri, Alejandro Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina
Materia
droplet digital PCR
SARS-CoV-2
pooled samples
ddPCR
COVID-19
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:11336/211592
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/211592
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network_name_str CONICET Digital (CONICET)
spelling Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCRHeckel, Sofía BelénPacini, AntonellaParedes, FrancoPetreli, Maria VictoriaPerez, MarilinaAdriani, NataliaIbarra, GuadalupeMenzella, Hugo GabrielColaneri, Alejandro CesarSesma, Julianadroplet digital PCRSARS-CoV-2pooled samplesddPCRCOVID-19https://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.Fil: Heckel, Sofía Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaFil: Pacini, Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaFil: Paredes, Franco. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Petreli, Maria Victoria. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Perez, Marilina. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; ArgentinaFil: Adriani, Natalia. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; ArgentinaFil: Ibarra, Guadalupe. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Menzella, Hugo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Colaneri, Alejandro Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaPublic Library of Science2022-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/211592Heckel, Sofía Belén; Pacini, Antonella; Paredes, Franco; Petreli, Maria Victoria; Perez, Marilina; et al.; Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR; Public Library of Science; Plos One; 17; 11-2022; 1-141932-6203CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0271860info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0271860info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:17:34Zoai:ri.conicet.gov.ar:11336/211592instacron: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:34982025-10-22 12:17:34.588CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
title Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
spellingShingle Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
Heckel, Sofía Belén
droplet digital PCR
SARS-CoV-2
pooled samples
ddPCR
COVID-19
title_short Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
title_full Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
title_fullStr Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
title_full_unstemmed Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
title_sort Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR
dc.creator.none.fl_str_mv Heckel, Sofía Belén
Pacini, Antonella
Paredes, Franco
Petreli, Maria Victoria
Perez, Marilina
Adriani, Natalia
Ibarra, Guadalupe
Menzella, Hugo Gabriel
Colaneri, Alejandro Cesar
Sesma, Juliana
author Heckel, Sofía Belén
author_facet Heckel, Sofía Belén
Pacini, Antonella
Paredes, Franco
Petreli, Maria Victoria
Perez, Marilina
Adriani, Natalia
Ibarra, Guadalupe
Menzella, Hugo Gabriel
Colaneri, Alejandro Cesar
Sesma, Juliana
author_role author
author2 Pacini, Antonella
Paredes, Franco
Petreli, Maria Victoria
Perez, Marilina
Adriani, Natalia
Ibarra, Guadalupe
Menzella, Hugo Gabriel
Colaneri, Alejandro Cesar
Sesma, Juliana
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv droplet digital PCR
SARS-CoV-2
pooled samples
ddPCR
COVID-19
topic droplet digital PCR
SARS-CoV-2
pooled samples
ddPCR
COVID-19
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.
Fil: Heckel, Sofía Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina
Fil: Pacini, Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina
Fil: Paredes, Franco. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; Argentina
Fil: Petreli, Maria Victoria. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; Argentina
Fil: Perez, Marilina. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina
Fil: Adriani, Natalia. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina
Fil: Ibarra, Guadalupe. Gobierno de la Provincia de Santa Fe. Hospital Provincial de Rosario.; Argentina. Universidad Nacional de Rosario; Argentina
Fil: Menzella, Hugo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina
Fil: Colaneri, Alejandro Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina
description Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.
publishDate 2022
dc.date.none.fl_str_mv 2022-11
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/11336/211592
Heckel, Sofía Belén; Pacini, Antonella; Paredes, Franco; Petreli, Maria Victoria; Perez, Marilina; et al.; Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR; Public Library of Science; Plos One; 17; 11-2022; 1-14
1932-6203
CONICET Digital
CONICET
url http://hdl.handle.net/11336/211592
identifier_str_mv Heckel, Sofía Belén; Pacini, Antonella; Paredes, Franco; Petreli, Maria Victoria; Perez, Marilina; et al.; Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR; Public Library of Science; Plos One; 17; 11-2022; 1-14
1932-6203
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://journals.plos.org/plosone/article?id=10.1371/journal.pone.0271860
info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0271860
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
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
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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