RESUMO
A conserved nucleic acid fragment of the nucleocapsid gene of Swine Transmissible Gastroenteritis Coronavirus (TGEV) was chosen as the target, six special primers were designed successfully. Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × SYBR greenI in the LAMP reaction. The assay established in this study was found to detect only the TGEV and no cross-reaction with other viruses, demonstrating its high specificity. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.
Assuntos
Gastroenterite Suína Transmissível/diagnóstico , Gastroenterite Suína Transmissível/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus da Gastroenterite Transmissível/isolamento & purificação , Virologia/métodos , Animais , Benzotiazóis , Sequência Conservada , Primers do DNA/genética , Diaminas , Nucleocapsídeo/genética , Compostos Orgânicos/metabolismo , Quinolinas , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Suínos , TemperaturaRESUMO
A rapid detection of foot-and-mouth disease virus (FMDV) was established by using reverse transcription loop-mediated isothermal amplification (RT-LAMP) method, meanwhile its specificity and sensitivity were assessed. The results showed that the FMDV RNA could be amplified by incubation at 65degrees C for only 1h using six primers designed based on FMDV polyprotein gene and the amplification products could be detected easily by naked-eye. There is no cross reaction with other virus such as SVDV, SFV and PPV by detecting their RNA samples. The detection limit of this method was found to be 10(-5) dilution of RNA sample which was 100-fold higher than that of PCR and 10-fold higher than real-time PCR.
