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首页> 外文期刊>Revista de salud animal >Standarization of a SYBR Green-I based real-time RT-PCR assay for detection of bluetongue virus in centinel animals
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Standarization of a SYBR Green-I based real-time RT-PCR assay for detection of bluetongue virus in centinel animals

机译：基于SYBR Green-I的实时RT-PCR分析的标准化，用于检测中心动物中的蓝舌病毒

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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.
机译：蓝舌病（BT）是反刍动物的一种昆虫传播的病毒性疾病，主要影响绵羊。该疾病是由呼肠孤病毒科中的Orbivirus病毒（BTV）引起的。迄今已鉴定出26种不同的病毒血清型。病毒的快速传播和临床体征的可变性值得发展迅速而准确的BTV检测方法，这些方法可以帮助控制疾病。基于SYBR Green的实时逆转录PCR（rRT-PCR）分析可能是快速检测动物血液中BTV基因组的理想方法。在这项研究中，描述了一种rRT-PCR与解链曲线分析的方法，并将其与OIE手册中报道的嵌套式逆转录聚合酶链反应（nRT-PCR）进行了比较。使用从十倍连续稀释的组织培养基病毒和血液中提取的BTV-4 RNA（从104.5 TCID50 / ml开始）评估两种方法的特异性和敏感性。在组织培养基中每种测试的检出限为100.5 TCID50 / ml，在血液中为101.5 TCID50 / ml（rRT-PCR）和100.5 TCID50 / ml（nRT-PCR）。熔解曲线分析显示，rRT-PCR产生具有特定熔解曲线（Tm = 84°C±0.5°C）且无非特异性扩增的扩增曲线。评估了小牛在受控条件下的临床样品的诊断敏感性。这些结果表明，基于SYBR Green I的实时PCR测定对于诊断受BTV感染的动物的BT具有快速，灵敏和同样的特异性。

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