Aplicação da técnica de RT-PCR in situ na detecção da co-infecção pelo Coronavírus grupo 3 (TCoV) e Astrovírus (TAstV-2) em perus com quadro agudo de enterite
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Data
2009-12-18
Autores
Rosa, Ana Carolina Guedes [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O presente estudo descreve o desenvolvimento e a aplicação da reação de transcrição reversa in situ em cadeia da polimerase (RT-PCR in situ), para detectar a co-infecção experimental de perus de 1-dia de idade com Coronavirus (TCoV) e Astrovirus (TAstV-2) isolados de casos clínicos no Brasil. A primeira etapa da reação consistiu na preparação específica de sondas de DNA biotiniladas homólogas ao gene da polimerase viral do TAstV-2 e da região 3'UTR do TCoV. Foram utilizados cortes histológicos de intestino correspondendo ás regiões do íleo, junção íleo-ceco e ceco para avaliar a reação de RT-PCR in situ. Para permeabilização tecidual uma digestão foi aplicada com 10 μg/μl proteinase K por 30 min. Na etapa de hibridização, as sondas de DNA homólogo às regiões genômicas virais ligadas à biotina foram diluídas na concentração de 2μg/μl na solução de hibridização e incubadas overnight à 42ºC. Em seguida, uma diluição ótima do anticorpo monoclonal anti-biotina acoplado a fosfatase alcalina e a peroxidase foram aplicados, para TAstV-2 e TCoV, respectivamente. O substratos diaminobenzidina 3,3 (DAB) e FastRed Ò foram utilizados para identificar a hibridização das regiões homólogas correspondentes ao TCoV e TAstV-2, respectivamente. A reação positiva foi visualizada por deposição de pigmentos vermelhos (TAstV-2) e marrom acastanhado (TCoV). Em relação à localização dos genes virais amplificados, foram confirmados nas células da base (células caliciformes) e ao longo das vilosidades intestinais principalmente no citoplasma dos enterócitos de forma difusa para ambos os vírus. Marcações positivas também foram evidenciadas na submucosa próximas às regiões com intensa congestão vascular...
This study describes the development and application of the reaction in situ reverse transcription polymerase chain reaction (RT-PCR in situ) to detect co-infection of turkeys to experimental 1-day-old with Coronavirus (TCoV) and Astrovirus ( TAstV-2) isolated from clinical cases in Brazil. The first step of the reaction has been to prepare specific biotinylated DNA probes homologous to the viral polymerase gene of TAstV-2 and the 3'UTR region of TCoV. We used histological sections of intestine corresponding to the regions of the ileum, ileum-cecum junction and cecum to evaluate the reaction of RT-PCR in situ. For permeabilization tissue digestion was applied with 10 g / uL proteinase K for 30 min. In step hybridization, DNA probes homologous to the viral genomic regions linked to biotin were diluted to the concentration of 2μg/μl in hybridization solution and incubated overnight to 42 ° C. Then, an optimal dilution of monoclonal anti-biotin coupled to alkaline phosphatase and peroxidase were applied to TAstV-2 and TCoV, respectively. 3.3 The substrate were used to identify the hybridization ofÒdiaminobenzidine (DAB) and FastRed homologous regions corresponding to TCoV and TAstV-2, respectively. The positive reaction was visualized by deposition of red pigment (TAstV-2) and brown brown (TCoV). Concerning the location of the amplified viral genes was confirmed by the base cells (goblet cells) and along the intestinal villi in the cytoplasm of enterocytes diffusely to both viruses. Tags positive were also demonstrated in the submucosa close to the areas with intense vascular congestion. In conclusion, the RT-PCR in situ standard in this study showed good ability to detect viral RNA promoting a desirable... (Complete abstract click electronic access below)
This study describes the development and application of the reaction in situ reverse transcription polymerase chain reaction (RT-PCR in situ) to detect co-infection of turkeys to experimental 1-day-old with Coronavirus (TCoV) and Astrovirus ( TAstV-2) isolated from clinical cases in Brazil. The first step of the reaction has been to prepare specific biotinylated DNA probes homologous to the viral polymerase gene of TAstV-2 and the 3'UTR region of TCoV. We used histological sections of intestine corresponding to the regions of the ileum, ileum-cecum junction and cecum to evaluate the reaction of RT-PCR in situ. For permeabilization tissue digestion was applied with 10 g / uL proteinase K for 30 min. In step hybridization, DNA probes homologous to the viral genomic regions linked to biotin were diluted to the concentration of 2μg/μl in hybridization solution and incubated overnight to 42 ° C. Then, an optimal dilution of monoclonal anti-biotin coupled to alkaline phosphatase and peroxidase were applied to TAstV-2 and TCoV, respectively. 3.3 The substrate were used to identify the hybridization ofÒdiaminobenzidine (DAB) and FastRed homologous regions corresponding to TCoV and TAstV-2, respectively. The positive reaction was visualized by deposition of red pigment (TAstV-2) and brown brown (TCoV). Concerning the location of the amplified viral genes was confirmed by the base cells (goblet cells) and along the intestinal villi in the cytoplasm of enterocytes diffusely to both viruses. Tags positive were also demonstrated in the submucosa close to the areas with intense vascular congestion. In conclusion, the RT-PCR in situ standard in this study showed good ability to detect viral RNA promoting a desirable... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Peru (Ave), Enterite, Coronavirus, Avastrovirus, Enteritis
Como citar
ROSA, Ana Carolina Guedes. Aplicação da técnica de RT-PCR in situ na detecção da co-infecção pelo Coronavírus grupo 3 (TCoV) e Astrovírus (TAstV-2) em perus com quadro agudo de enterite. 2009. 50 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Odontologia e Curso de Medicina Veterinária, 2009.