Standarization of a SYBR Green-I based real-time RT-PCR assay for detection of bluetongue virus in centinel animals

[摘要] Bluetongue (BT) is an insect-transmitted viral disease of ruminant species that primarily affects sheep. This disease is caused by a virus (BTV) of the genus Orbivirus within the family Reoviridae. Twenty-six distinct serotypes of the virus have been identified to date. The rapid dissemination of the virus and the variability of the clinical signs merit the development of swift and accurate BTV detection methods, which can assist in disease control. SYBR Green-based real-time reverse transcription-PCR (rRT-PCR) assay may be an ideal method to detect rapidly the BTV genome in the blood of animals. In this study, a rRT-PCR coupled to melting curve analysis was described and compared with the nested reverse transcription-polymerase chain reaction (nRT-PCR) reported in the OIE Manual. The specificity and sensitivity of both methods were evaluated using BTV-4 RNA extracted from tenfold serially diluted tissue culture medium virus and blood (starting from 104.5 TCID50/ml). The detection limit of each test in tissue culture medium was 100.5 TCID50/ml and in blood was 101.5 TCID50/ml (rRT-PCR) and 100.5 TCID50/ml (nRT-PCR). Melting curve analysis showed that the rRT-PCR yielded curves of amplification with specific melting curves (Tm = 84°C ± 0.5°C) and absence of non-specific amplifications. The diagnostic sensibility of clinical samples from a calf under controlled conditions was evaluated. These results showed that the SYBR Green I-based real-time PCR assay is rapid, sensitive, and equally specific in the diagnosis of BT in BTV-affected animals.

[学科分类] 兽医学

[关键词] bluetongue virus;nested RT-PCR;SYBR Green I-based real-time RT-PCR assay