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dc.contributor.author
Tsukamoto, Kenji
dc.contributor.author
Panei, Carlos Javier
dc.contributor.author
Shishido, Makiko
dc.contributor.author
Noguchi, Daigo
dc.contributor.author
Pearce, John
dc.contributor.author
Kang, Hyun Mi
dc.contributor.author
Jeong, Ok Mi
dc.contributor.author
Lee, Youn Jeong
dc.contributor.author
Nakanishi, Koji
dc.contributor.author
Ashizawa, Takayoshi
dc.date.available
2017-01-13T18:56:25Z
dc.date.issued
2012-01
dc.identifier.citation
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
dc.identifier.issn
0095-1137
dc.identifier.uri
http://hdl.handle.net/11336/11320
dc.description.abstract
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.
dc.format
application/pdf
dc.language.iso
eng
dc.publisher
American Society For Microbiology
dc.rights
info:eu-repo/semantics/openAccess
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.subject
Aivs
dc.subject
Sybr-Green
dc.subject.classification
Ciencias Veterinarias
dc.subject.classification
Ciencias Veterinarias
dc.subject.classification
CIENCIAS AGRÍCOLAS
dc.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
dc.type
info:eu-repo/semantics/article
dc.type
info:ar-repo/semantics/artículo
dc.type
info:eu-repo/semantics/publishedVersion
dc.date.updated
2017-01-13T13:59:52Z
dc.identifier.eissn
1098-660X
dc.journal.volume
50
dc.journal.number
1
dc.journal.pagination
37-45
dc.journal.pais
Estados Unidos
dc.journal.ciudad
Washington DC
dc.description.fil
Fil: Tsukamoto, Kenji . National Institute of Animal Health; Japón
dc.description.fil
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
dc.description.fil
Fil: Shishido, Makiko . National Institute of Animal Health; Japón
dc.description.fil
Fil: Noguchi, Daigo . National Institute of Animal Health; Japón
dc.description.fil
Fil: Pearce, John. Alaska Science Center; Estados Unidos
dc.description.fil
Fil: Kang, Hyun Mi. National Veterinary Research and Quarantine Service; Corea del Sur
dc.description.fil
Fil: Jeong, Ok Mi . National Veterinary Research and Quarantine Service; Corea del Sur
dc.description.fil
Fil: Lee, Youn Jeong . National Veterinary Research and Quarantine Service; Corea del Sur
dc.description.fil
Fil: Nakanishi, Koji . Livestock Hygiene Service Center of Shiga
Prefecture; Japón
dc.description.fil
Fil: Ashizawa, Takayoshi . Tyuou Livestock Hygiene Service Center of Chiba Prefecture; Japón
dc.journal.title
Journal Of Clinical Microbiology
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.1128/JCM.01195-11
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/http://jcm.asm.org/content/50/1/37
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