Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples

Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious vi...

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Autores: Randazzo, Walter, Khezri, Mohammad, Ollivier, Joanna, Le Guyader, Françoise S., Rodríguez Díaz, Jesús, Aznar, Rosa, Sánchez Moragas, Gloria
Formato: artículo
Estado:Versión aceptada para publicación
Fecha de publicación:2017
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/158997
Acesso em linha:http://hdl.handle.net/10261/158997
Access Level:acceso abierto
Palavra-chave:Viability PCR
Norovirus
Shellfish
Intercalating dyes
Sewage
RT-qPCR
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spelling Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samplesRandazzo, WalterKhezri, MohammadOllivier, JoannaLe Guyader, Françoise S.Rodríguez Díaz, JesúsAznar, RosaSánchez Moragas, GloriaViability PCRNorovirusShellfishIntercalating dyesSewageRT-qPCRShellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30 min incubation with 100 μM of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99 °C for 5 min) HuNoV GI and GII. This pretreatment reduced the signal of thermally-inactivated HuNoV GI in cockles and HuNoV GII in mussels by > 3 log. Additionally, this pretreatment reduced the signal of thermally-inactivated HuNoV GI and GII between 1 and 1.5 log in oysters. Thermal inactivation of HuNoV GI and GII in PBS, sewage and bioaccumulated oysters was also evaluated by the PMAxx-Triton pretreatment. Results showed significant differences between reductions observed in the control and PMAxx-treated samples in PBS following treatment at 72 and 95 °C for 15 min. In sewage, the RT-qPCR signal of HuNoV GI was completely removed by the PMAxx pretreatment after heating at 72 and 95 °C, while the RT-qPCR signal for HuNoV GII was completely eliminated only at 95 °C. Finally, the PMAxx-Triton pretreatment was applied to naturally contaminated sewage and oysters, resulting in most of the HuNoV genomes quantified in sewage and oyster samples (12 out of 17) corresponding to undamaged capsids. Although this procedure may still overestimate infectivity, the PMAxx-Triton pretreatment represents a step forward to better interpret the quantification of intact HuNoVs in complex matrices, such as sewage and shellfish, and it could certainly be included in the procedures based on RT-qPCR.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (RYC-2012-09950 and RYC-2013-12442) and the Spanish National Institute for Agriculture and Food Research and Technology (INIA) co-financed by the European Social Fund (Project RTA2014-00024-C03). Financial support has been co-sponsored by the European Regional Development Fund (FEDER). GS and JRD were supported by the “Ramón y Cajal” Young Investigator.Peer reviewedElsevierMinisterio de Economía y Competitividad (España)CSIC - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)European CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201820182017info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionhttp://hdl.handle.net/10261/158997reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/RTA2014-00024-C03https://doi.org/10.1016/j.ijfoodmicro.2017.11.011Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1589972026-05-22T06:33:51Z
dc.title.none.fl_str_mv Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
title Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
spellingShingle Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
Randazzo, Walter
Viability PCR
Norovirus
Shellfish
Intercalating dyes
Sewage
RT-qPCR
title_short Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
title_full Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
title_fullStr Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
title_full_unstemmed Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
title_sort Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
dc.creator.none.fl_str_mv Randazzo, Walter
Khezri, Mohammad
Ollivier, Joanna
Le Guyader, Françoise S.
Rodríguez Díaz, Jesús
Aznar, Rosa
Sánchez Moragas, Gloria
author Randazzo, Walter
author_facet Randazzo, Walter
Khezri, Mohammad
Ollivier, Joanna
Le Guyader, Françoise S.
Rodríguez Díaz, Jesús
Aznar, Rosa
Sánchez Moragas, Gloria
author_role author
author2 Khezri, Mohammad
Ollivier, Joanna
Le Guyader, Françoise S.
Rodríguez Díaz, Jesús
Aznar, Rosa
Sánchez Moragas, Gloria
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
CSIC - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Viability PCR
Norovirus
Shellfish
Intercalating dyes
Sewage
RT-qPCR
topic Viability PCR
Norovirus
Shellfish
Intercalating dyes
Sewage
RT-qPCR
description Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30 min incubation with 100 μM of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99 °C for 5 min) HuNoV GI and GII. This pretreatment reduced the signal of thermally-inactivated HuNoV GI in cockles and HuNoV GII in mussels by > 3 log. Additionally, this pretreatment reduced the signal of thermally-inactivated HuNoV GI and GII between 1 and 1.5 log in oysters. Thermal inactivation of HuNoV GI and GII in PBS, sewage and bioaccumulated oysters was also evaluated by the PMAxx-Triton pretreatment. Results showed significant differences between reductions observed in the control and PMAxx-treated samples in PBS following treatment at 72 and 95 °C for 15 min. In sewage, the RT-qPCR signal of HuNoV GI was completely removed by the PMAxx pretreatment after heating at 72 and 95 °C, while the RT-qPCR signal for HuNoV GII was completely eliminated only at 95 °C. Finally, the PMAxx-Triton pretreatment was applied to naturally contaminated sewage and oysters, resulting in most of the HuNoV genomes quantified in sewage and oyster samples (12 out of 17) corresponding to undamaged capsids. Although this procedure may still overestimate infectivity, the PMAxx-Triton pretreatment represents a step forward to better interpret the quantification of intact HuNoVs in complex matrices, such as sewage and shellfish, and it could certainly be included in the procedures based on RT-qPCR.
publishDate 2017
dc.date.none.fl_str_mv 2017
2018
2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/158997
url http://hdl.handle.net/10261/158997
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/RTA2014-00024-C03
https://doi.org/10.1016/j.ijfoodmicro.2017.11.011
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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