SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard ser...

Autores
Tsukamoto, Kenji; Panei, Carlos Javier; Shishido, Makiko; Noguchi, Daigo; Pearce, John; Kang, Hyun Mi; Jeong, Ok Mi; Lee, Youn Jeong; Nakanishi, Koji; Ashizawa, Takayoshi
Año de publicación
2012
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 101.5, 102.3, and 103.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.
Fil: Tsukamoto, Kenji . National Institute of Animal Health; Japón
Fil: Panei, Carlos Javier. National Institute of Animal Health; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Estudios Parasitológicos y de Vectores (i); Argentina
Fil: Shishido, Makiko . National Institute of Animal Health; Japón
Fil: Noguchi, Daigo . National Institute of Animal Health; Japón
Fil: Pearce, John. Alaska Science Center; Estados Unidos
Fil: Kang, Hyun Mi. National Veterinary Research and Quarantine Service; Corea del Sur
Fil: Jeong, Ok Mi . National Veterinary Research and Quarantine Service; Corea del Sur
Fil: Lee, Youn Jeong . National Veterinary Research and Quarantine Service; Corea del Sur
Fil: Nakanishi, Koji . Livestock Hygiene Service Center of Shiga Prefecture; Japón
Fil: Ashizawa, Takayoshi . Tyuou Livestock Hygiene Service Center of Chiba Prefecture; Japón
Materia
Aivs
Sybr-Green
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:11336/11320
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/11320
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping testsTsukamoto, Kenji Panei, Carlos JavierShishido, Makiko Noguchi, Daigo Pearce, JohnKang, Hyun MiJeong, Ok Mi Lee, Youn Jeong Nakanishi, Koji Ashizawa, Takayoshi AivsSybr-Greenhttps://purl.org/becyt/ford/4.3https://purl.org/becyt/ford/4Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 101.5, 102.3, and 103.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.Fil: Tsukamoto, Kenji . National Institute of Animal Health; JapónFil: Panei, Carlos Javier. National Institute of Animal Health; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Estudios Parasitológicos y de Vectores (i); ArgentinaFil: Shishido, Makiko . National Institute of Animal Health; JapónFil: Noguchi, Daigo . National Institute of Animal Health; JapónFil: Pearce, John. Alaska Science Center; Estados UnidosFil: Kang, Hyun Mi. National Veterinary Research and Quarantine Service; Corea del SurFil: Jeong, Ok Mi . National Veterinary Research and Quarantine Service; Corea del SurFil: Lee, Youn Jeong . National Veterinary Research and Quarantine Service; Corea del SurFil: Nakanishi, Koji . Livestock Hygiene Service Center of Shiga Prefecture; JapónFil: Ashizawa, Takayoshi . Tyuou Livestock Hygiene Service Center of Chiba Prefecture; JapónAmerican Society For Microbiology2012-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/11320Tsukamoto, Kenji ; Panei, Carlos Javier; Shishido, Makiko ; Noguchi, Daigo ; Pearce, John; et al.; SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests; American Society For Microbiology; Journal Of Clinical Microbiology; 50; 1; 1-2012; 37-450095-11371098-660Xenginfo:eu-repo/semantics/altIdentifier/doi/10.1128/JCM.01195-11info:eu-repo/semantics/altIdentifier/url/http://jcm.asm.org/content/50/1/37info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:01:30Zoai:ri.conicet.gov.ar:11336/11320instacron: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:34982025-09-10 13:01:30.29CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
title SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
spellingShingle SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
Tsukamoto, Kenji
Aivs
Sybr-Green
title_short SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
title_full SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
title_fullStr SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
title_full_unstemmed SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
title_sort SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests
dc.creator.none.fl_str_mv Tsukamoto, Kenji
Panei, Carlos Javier
Shishido, Makiko
Noguchi, Daigo
Pearce, John
Kang, Hyun Mi
Jeong, Ok Mi
Lee, Youn Jeong
Nakanishi, Koji
Ashizawa, Takayoshi
author Tsukamoto, Kenji
author_facet Tsukamoto, Kenji
Panei, Carlos Javier
Shishido, Makiko
Noguchi, Daigo
Pearce, John
Kang, Hyun Mi
Jeong, Ok Mi
Lee, Youn Jeong
Nakanishi, Koji
Ashizawa, Takayoshi
author_role author
author2 Panei, Carlos Javier
Shishido, Makiko
Noguchi, Daigo
Pearce, John
Kang, Hyun Mi
Jeong, Ok Mi
Lee, Youn Jeong
Nakanishi, Koji
Ashizawa, Takayoshi
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Aivs
Sybr-Green
topic Aivs
Sybr-Green
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.3
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 101.5, 102.3, and 103.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.
Fil: Tsukamoto, Kenji . National Institute of Animal Health; Japón
Fil: Panei, Carlos Javier. National Institute of Animal Health; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Estudios Parasitológicos y de Vectores (i); Argentina
Fil: Shishido, Makiko . National Institute of Animal Health; Japón
Fil: Noguchi, Daigo . National Institute of Animal Health; Japón
Fil: Pearce, John. Alaska Science Center; Estados Unidos
Fil: Kang, Hyun Mi. National Veterinary Research and Quarantine Service; Corea del Sur
Fil: Jeong, Ok Mi . National Veterinary Research and Quarantine Service; Corea del Sur
Fil: Lee, Youn Jeong . National Veterinary Research and Quarantine Service; Corea del Sur
Fil: Nakanishi, Koji . Livestock Hygiene Service Center of Shiga Prefecture; Japón
Fil: Ashizawa, Takayoshi . Tyuou Livestock Hygiene Service Center of Chiba Prefecture; Japón
description Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 101.5, 102.3, and 103.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.
publishDate 2012
dc.date.none.fl_str_mv 2012-01
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/11336/11320
Tsukamoto, Kenji ; Panei, Carlos Javier; Shishido, Makiko ; Noguchi, Daigo ; Pearce, John; et al.; SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests; American Society For Microbiology; Journal Of Clinical Microbiology; 50; 1; 1-2012; 37-45
0095-1137
1098-660X
url http://hdl.handle.net/11336/11320
identifier_str_mv Tsukamoto, Kenji ; Panei, Carlos Javier; Shishido, Makiko ; Noguchi, Daigo ; Pearce, John; et al.; SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all Hemagglutinin and Neuraminidase genes of Avian Influenza viruses and comparison to standard serological subtyping tests; American Society For Microbiology; Journal Of Clinical Microbiology; 50; 1; 1-2012; 37-45
0095-1137
1098-660X
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1128/JCM.01195-11
info:eu-repo/semantics/altIdentifier/url/http://jcm.asm.org/content/50/1/37
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 American Society For Microbiology
publisher.none.fl_str_mv American Society For Microbiology
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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score 12.993085

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