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First application of a droplet digital PCR to detect Toxoplasma gondii in mussels

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and releas...

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Autores principales: Mancusi, Andrea, Proroga, Yolande T. R., Giordano, Angela, Girardi, Santa, D’Orilia, Francescantonio, Pinto, Renato, Sarnelli, Paolo, Rinaldi, Laura, Capuano, Federico, Maurelli, Maria Paola
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10514481/
https://www.ncbi.nlm.nih.gov/pubmed/37744920
http://dx.doi.org/10.3389/fmicb.2023.1238689
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author Mancusi, Andrea
Proroga, Yolande T. R.
Giordano, Angela
Girardi, Santa
D’Orilia, Francescantonio
Pinto, Renato
Sarnelli, Paolo
Rinaldi, Laura
Capuano, Federico
Maurelli, Maria Paola
author_facet Mancusi, Andrea
Proroga, Yolande T. R.
Giordano, Angela
Girardi, Santa
D’Orilia, Francescantonio
Pinto, Renato
Sarnelli, Paolo
Rinaldi, Laura
Capuano, Federico
Maurelli, Maria Paola
author_sort Mancusi, Andrea
collection PubMed
description Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/μL, 800 gc/μL, 80 gc/μL, 8 gc/μL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/μL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3–99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7–25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans.
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spelling pubmed-105144812023-09-23 First application of a droplet digital PCR to detect Toxoplasma gondii in mussels Mancusi, Andrea Proroga, Yolande T. R. Giordano, Angela Girardi, Santa D’Orilia, Francescantonio Pinto, Renato Sarnelli, Paolo Rinaldi, Laura Capuano, Federico Maurelli, Maria Paola Front Microbiol Microbiology Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/μL, 800 gc/μL, 80 gc/μL, 8 gc/μL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/μL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3–99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7–25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans. Frontiers Media S.A. 2023-09-08 /pmc/articles/PMC10514481/ /pubmed/37744920 http://dx.doi.org/10.3389/fmicb.2023.1238689 Text en Copyright © 2023 Mancusi, Proroga, Giordano, Girardi, D’Orilia, Pinto, Sarnelli, Rinaldi, Capuano and Maurelli. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Mancusi, Andrea
Proroga, Yolande T. R.
Giordano, Angela
Girardi, Santa
D’Orilia, Francescantonio
Pinto, Renato
Sarnelli, Paolo
Rinaldi, Laura
Capuano, Federico
Maurelli, Maria Paola
First application of a droplet digital PCR to detect Toxoplasma gondii in mussels
title First application of a droplet digital PCR to detect Toxoplasma gondii in mussels
title_full First application of a droplet digital PCR to detect Toxoplasma gondii in mussels
title_fullStr First application of a droplet digital PCR to detect Toxoplasma gondii in mussels
title_full_unstemmed First application of a droplet digital PCR to detect Toxoplasma gondii in mussels
title_short First application of a droplet digital PCR to detect Toxoplasma gondii in mussels
title_sort first application of a droplet digital pcr to detect toxoplasma gondii in mussels
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10514481/
https://www.ncbi.nlm.nih.gov/pubmed/37744920
http://dx.doi.org/10.3389/fmicb.2023.1238689
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