Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1590/S1984-29612022017 http://hdl.handle.net/11449/234304 |
Resumo: | The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples. |
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Repositório Institucional da UNESP |
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spelling |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samplesThe aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.Faculdade de Medicina Veterinária Universidade Estadual Paulista - UNESPLaboratório de Parasitologia Animal Instituto BiológicoFaculdade de Medicina Veterinária Universidade Estadual Paulista - UNESPUniversidade Estadual Paulista (UNESP)Instituto BiológicoSantana, Bruna Nicoleti [UNESP]Ferrari, Elis Domingos [UNESP]Nakamura, Alex Akira [UNESP]Silva, Giane Serafim daMeireles, Marcelo Vasconcelos [UNESP]2022-05-01T15:46:17Z2022-05-01T15:46:17Z2022-01-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlee000522http://dx.doi.org/10.1590/S1984-29612022017Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria, v. 31, n. 1, p. e000522-, 2022.1984-2961http://hdl.handle.net/11449/23430410.1590/S1984-296120220172-s2.0-85126890297Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengRevista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinariainfo:eu-repo/semantics/openAccess2024-09-04T18:04:25Zoai:repositorio.unesp.br:11449/234304Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestrepositoriounesp@unesp.bropendoar:29462024-09-04T18:04:25Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples |
title |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples |
spellingShingle |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples Santana, Bruna Nicoleti [UNESP] |
title_short |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples |
title_full |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples |
title_fullStr |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples |
title_full_unstemmed |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples |
title_sort |
Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples |
author |
Santana, Bruna Nicoleti [UNESP] |
author_facet |
Santana, Bruna Nicoleti [UNESP] Ferrari, Elis Domingos [UNESP] Nakamura, Alex Akira [UNESP] Silva, Giane Serafim da Meireles, Marcelo Vasconcelos [UNESP] |
author_role |
author |
author2 |
Ferrari, Elis Domingos [UNESP] Nakamura, Alex Akira [UNESP] Silva, Giane Serafim da Meireles, Marcelo Vasconcelos [UNESP] |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (UNESP) Instituto Biológico |
dc.contributor.author.fl_str_mv |
Santana, Bruna Nicoleti [UNESP] Ferrari, Elis Domingos [UNESP] Nakamura, Alex Akira [UNESP] Silva, Giane Serafim da Meireles, Marcelo Vasconcelos [UNESP] |
description |
The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-05-01T15:46:17Z 2022-05-01T15:46:17Z 2022-01-01 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1590/S1984-29612022017 Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria, v. 31, n. 1, p. e000522-, 2022. 1984-2961 http://hdl.handle.net/11449/234304 10.1590/S1984-29612022017 2-s2.0-85126890297 |
url |
http://dx.doi.org/10.1590/S1984-29612022017 http://hdl.handle.net/11449/234304 |
identifier_str_mv |
Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria, v. 31, n. 1, p. e000522-, 2022. 1984-2961 10.1590/S1984-29612022017 2-s2.0-85126890297 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
e000522 |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
repositoriounesp@unesp.br |
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1810021427137478656 |