Detecção e diferenciação do vírus da bronquite infecciosa pela técnica de imunocaptura-RT-PCR E RFLP
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2007-07-06
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Universidade Estadual Paulista (Unesp)
Resumo
Nesse estudo foi desenvolvido e aplicado o procedimento de imunocaptura para realizar a reação de transcrição reversa seguida pela reação em cadeia da polimerase (IC-RT-PCR) a fim de ser amplificada uma região 5'_ proximal do gene 81 do vírus da bronquite infecciosa (VBI) com 228 pb e de forma a fazer a detecção e a diferenciação desse vírus, comparando-se os resultados dessa metodologia com os obtidos na técnica convencional de RT-PCR. Todas as 11 estirpes do VBI testadas foram amplificadas pelas duas técnicas moleculares, enquanto que nenhum dos vírus heterólogos (Pneumovírus Aviário do grupo A e B, Vírus da Doença de Gumboro e o Vírus da Doença de Newcastle) ensaiados levaram a amplificação específica do gene 81. O limiar de detecção para a técnica de IC-RT-PCR foi idêntico ao do método de RT-PCR e correspondeu a 102.8 Doses Infectantes Embrionárias 50%. Para um total de 35 amostras de tecidos do trato respiratório testadas e provenientes de aves infectadas experimentalmente, 32 foram positivas pela técnica de IC-RT-PCR e também pela RT-PCR convencional, enquanto que o isolamento viral foi obtido para 22 dessas amostras. A análise do produto amplificado do gene 81 na IC-RT-PCR através da técnica de RFLP (restriction fragment length polymorphism), com as enzimas Alul e Mboll, permitiu a diferenciação das 5 estirpes de referências e dos 6 isolados de campo analisados em 4 diferentes genótipos, correspondentes às estirpes de referência M41, Connecticut, ou 8E-17, ou a um isolado de campo. Portanto, a técnica de IC¬RT -PCR demonstrou um grande potencial para ser aplicada no diagnóstico direto do VBI, apresentando vantagens sobre a técnica convencional de RT-PCR, as quais foram derivadas da combinação da especificidade da imunocaptura em fase sólida com a sensibilidade da reação de PCR, o que pode proporcionar ganho de tempo e menor custo para a manipulação de um maior número de amostras.
In this study, the immunocapture procedure followed by reverse transcription and polymerase chain reaction (IC-RT-PCR) technique was standardized and applied for the amplification of 5'- proximal pari of 81 gene of infectious bronchitis virus (IBV) in infected f1uid or tissue samples, which were collected from embryonating chicken eggs or experimentally infected birds. The results of this technique were compared with those obtained in conventional RT-PCR. Ali eleven IBV strains tested were amplified, while none of the heterologous avian viral pathogens (Groups A and B Avian Pneumovirus, Newcastle Disease Virus and Gumboro Disease Vírus) gave positive results. The limit of detection for IC-RT¬PCR was identical to the common RT-PCR and corresponded to 102.8 50% embryonic infectious doses. Thirty two out of thirty five respiratory tissue samples collected from experimentally infected chickens were positive by both molecular techniques (IC-RT-PCR I common RT-PCR), whereas the vírus isolation test detected IBV in twenty two of these samples. The AluL and Mobll RFLP analysis of the 228 bp amplicon generated from IC-RT-PCR led to the discrimination of the 5 reference strains and 6 field IBV isolates in four genotypes; which were associated to the M41, to Connecticut, ar to 8E-17 strain, or to a field isolate. Therefore, the IC-RT-PCR demonstrated in this study a high potential for the application in the direct diagnosis of IBV and has relevant advantages over the conventional RT¬PCR, because it combines the specificity of the immunocapture in a solid phase with the sensitivity of the PCR, providing simplicity, rapidity and low cost for the manipulation of a high number of samples.
In this study, the immunocapture procedure followed by reverse transcription and polymerase chain reaction (IC-RT-PCR) technique was standardized and applied for the amplification of 5'- proximal pari of 81 gene of infectious bronchitis virus (IBV) in infected f1uid or tissue samples, which were collected from embryonating chicken eggs or experimentally infected birds. The results of this technique were compared with those obtained in conventional RT-PCR. Ali eleven IBV strains tested were amplified, while none of the heterologous avian viral pathogens (Groups A and B Avian Pneumovirus, Newcastle Disease Virus and Gumboro Disease Vírus) gave positive results. The limit of detection for IC-RT¬PCR was identical to the common RT-PCR and corresponded to 102.8 50% embryonic infectious doses. Thirty two out of thirty five respiratory tissue samples collected from experimentally infected chickens were positive by both molecular techniques (IC-RT-PCR I common RT-PCR), whereas the vírus isolation test detected IBV in twenty two of these samples. The AluL and Mobll RFLP analysis of the 228 bp amplicon generated from IC-RT-PCR led to the discrimination of the 5 reference strains and 6 field IBV isolates in four genotypes; which were associated to the M41, to Connecticut, ar to 8E-17 strain, or to a field isolate. Therefore, the IC-RT-PCR demonstrated in this study a high potential for the application in the direct diagnosis of IBV and has relevant advantages over the conventional RT¬PCR, because it combines the specificity of the immunocapture in a solid phase with the sensitivity of the PCR, providing simplicity, rapidity and low cost for the manipulation of a high number of samples.
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Palavras-chave
Frango de corte, Bronquite infecciosa em ave domestica, Diagnóstico molecular, Infectious bronchitis virus, Molecular diagnosis, Immunocapture, Variant characterization
Como citar
PIZA, Vanessa Mirabelli Toledo. Detecção e diferenciação do vírus da bronquite infecciosa pela técnica de imunocaptura-RT-PCR E RFLP. 2007. xv, 61 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2007.