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Master's Dissertation
DOI
10.11606/D.10.2019.tde-17102018-084452
Document
Author
Full name
Ruy Diego Chacon Villanueva
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2018
Supervisor
Committee
Ferreira, Antônio José Piantino (President)
Dagli, Maria Lucia Zaidan
Sesti, Luiz
Title in Portuguese
Desenvolvimento de reações de semi-nested PCR para o diagnóstico do vírus da Bronquite Infecciosa das Aves e sequenciamento de amostras brasileiras
Keywords in Portuguese
BR-I
Bronquite Infecciosa das Aves
Massachusetts
Proteína da espícula
Semi-Nested PCR
Abstract in Portuguese
A Bronquite Infecciosa das Aves (BIG) é uma das doenças respiratórias aviárias de maior impacto na avicultura mundial. No Brasil, as estirpes BR-I (GI-11) e Massachusetts (GI-1) são as mais prevalentes nos planteis avícolas. O presente estudo teve como objetivos desenvolver reações de semi-nested PCR para o diagnóstico das estirpes BR-I e Mass, em amostras brasileiras obtidas durante o período de 2016 e 2017. Foram desenvolvidas duas reações de semi-nested PCR tendo como alvo a subunidade 1 do gene S, específicas para as estirpes BR-I e Mass. O limiar de detecção foi de 104 cópias de DNA/µL nas duas reações (3,76fg/µL na reação exclusiva de BR-I; e 5,58fg/µL e 5,57fg/µL na reação duplex de BR-I e Mass, respectivamente). Posteriormente, foram avaliados 572 pools de órgãos procedentes das 5 regiões do Brasil. Dentre estas amostras, 62,24% foram positivas para Coronavírus, sendo o alvo desta reação a região 3UTR. A reação de semi-nested PCR específica detectou a estirpe BR-I em 84,83% das amostras positivas para Coronavírus. A reação de semi-nested PCR duplex detectou 65,44% das amostras positivas para a estirpe BR-I; 7,35% positivas para a estirpe Mass e co-infecção da estirpe BR-I com Mass em 17,65% das amostras. Após a análise dos controles positivos (vacinas Mass e BR-I) no BLASTn, do resultado do sequenciamento dos produtos de PCR, da análise filogenética, da similaridade de nucleotídeos e a dedução em aminoácidos, foi confirmado o agrupamento esperado das sequências detectadas pelas reações PCR dirigidas para a estirpe BR-I ou Mass. Estes resultados confirmaram a presença predominante da estirpe BR-I, e em menor número, da estirpe Mass nos planteis avícolas do Brasil. As reações desenvolvidas no presente estudo serão valiosas no diagnóstico e na monitoria da doença.
Title in English
Development of hemi-nested PCR reactions for the diagnosis of Avian Infectious Bronchitis virus and brazilian samples sequencing
Keywords in English
Avian Infectious Bronchitis
BR-I
Hemi-Nested PCR
Massachusetts
Spike protein
Abstract in English
Avian Infectious Bronchitis (IB) is one of the avian respiratory diseases with the greatest impact on poultry farming worldwide. In Brazil, strains BR-I (GI-11) and Massachusetts (GI-1) are the most prevalent in poultry flocks. The present study aimed to develop semi-nested PCR reactions for the diagnosis of IBV BR-I and Mass strains, in Brazilian samples obtained during the period of 2016 and 2017. Two semi-nested PCR reactions targeting the 1 subunit of the S gene were developed, specific for BR-I and Mass strains. The detection threshold was 104 copies of DNA/µL in both reactions (3,76fg/µL in the exclusive BR-I reaction; and 5,58fg/µL and 5,57fg/µL in the duplex reaction of BR-I and Mass, respectively). Subsequently, 572 organ pools from the 5 regions of Brazil were evaluated. Among these samples, 62,24% were positive for Coronavirus, being the target of this reaction the 3UTR region. The specific semi-nested PCR reaction detected the BR-I strain in 84,83% of the Coronavirus positive samples. The duplex semi-nested PCR reaction detected 65,44% of the samples positive for the BR-I strain, 7,35% positive for Mass strain, and co-infection of the BR-I and Mass strain in 17,65% of the samples. After the analysis of the positive controls (Mass and BR-I vaccines) in BLASTn, the result of the sequencing of the PCR products, phylogenetic analysis, nucleotide similarity and amino acid deduction, was confirmed the expected clustering of the sequences detected by the PCR reactions directed to BR-I and Mass strains. These results confirm the predominant presence of the BR-I strain, and to a lesser extent, the Mass strain in Brazilian poultry flocks. The reactions developed in the present study will be valuabe in the diagnosis and monitoring of the disease.
 
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Release Date
2020-10-29
Publishing Date
2019-01-09
 
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