Desenvolvimento de PCR convencional e em tempo real para o Vírus da Língua Azul

Abstract

The Bluetongue (BT) is a non-contagious infectious disease that has important socio-economic impacts of restrictions on import and / or by direct losses in cattle infected with bluetongue virus (BTV). The BTV is a member of the family Reoviridae, genus Orbivirus, transmitted by an arthropod vector of the genus Culicoides. The BTV can affect domestic and wild ruminants, but clinical disease is especially severe in sheep and some species of deer. To date, 24 serotypes are reported. The BTV is endemic in many parts of the world, including Africa and parts of theU.S., Australia and Asia. This study aimed to standardize the technique of polymerase chain reaction (PCR) and conventional real-time detection of BTV. After RNA extraction was performed followed by a reverse transcription PCR and real-time using specific primers for the gene that encodes the nonstructural protein NS3 of BTV, producing a fragment of 256pb. Standardized tests have been performed with semen and blood samples from cattle experimentally infected with BTV-4. The results showed amplification of the fragment of expected size (256pb), validating thus the protocol developed for conventional PCR and realtime PCR. From the conventional PCR detected an analytical sensitivity of 30fg, using cDNA of the suspension of BTV-4 and 250 g of nucleic acid (10-18ag), using plasmid DNA as template. The PCR results in real time, according to the standard curve, determination coefficients (R2) around 0.998 and slope of -3.528, indicating an amplification efficiency of about 92%, being reliable for the detection of BTV. The conventional PCR and real-time PCR were also tested on blood samples of cattle, goats and sheep from a property of MG with suspected erosive disease. All tests here have pointed out that the real-time PCR was able to detect BTV with a satisfactory sensitivity and specificity can be used in experimentally andnaturally infected samples. The PCR showed a lower sensitivity in detecting the virus in field sample.