Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of bovine ephemeral fever virus
Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of bovine ephemeral fever virus
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DOI：
10.1016/j.jviromet.2005.05.009
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发表时间：
2005-10-01
影响因子：
3.1
通讯作者：
Liu, HJ
中科院分区：
文献类型：
--
作者：
Hsieh, YC;Chen, SH;Liu, HJ
开发了一种快速、灵敏且特异的检测方法 RAPID-BAP 检测方法来检测和定量牛短暂热病毒 (BEFV) 的 G 蛋白编码基因。这项新技术使用巢式 PCR 和基于磁珠的 DNA 探测分析。检查了测定的最佳条件。通过应用巢式 PCR，该测定可以检测到至少 1 个拷贝/μl 的 BEFV 质粒 DNA。最佳杂交条件为 50 摄氏度，在 5x SSC 和 0.5% SDS 中孵育 20 分钟，可以清楚地区分阴性对照和阳性对照。该测定也具有高度特异性，因为所有阴性对照均未显示任何阳性检测。 RAPID-BAP法、实时RT-PCR法和常规RT-PCR法检测34份疑似BEFV感染的临床血样的诊断灵敏度分别为72.73%、36.36%和18.18%。结果表明，本研究开发的 RAPID-BAP 检测比传统 RT-PCR 和实时 RT-PCR 检测 BEFV 检测更灵敏。新型 RAPID-BAP 检测是一种出色的诊断工具，具有高灵敏度、特异性和快速周转时间。 (c) 2005 Elsevier B.V. 保留所有紧身衣。
A rapid, sensitive, and specific assay, RAPID-BAP assay, was developed to detect and quantify the G protein-encoding gene of bovine ephemeral fever virus (BEFV). This new technique uses a nested PCR and magnetic bead-based DNA probing assay. The optimal conditions for the assay were examined. By applying a nested PCR, a minimum of 1 copy/mu l of the BEFV plasmid DNA could be detected by the assay. The optimal hybridization conditions at 50 degrees C in 5x SSC and 0.5% SDS with a 20-min incubation allowed clear discrimination between negative and positive controls. The assay was also highly specific as all negative controls failed to show any positive detection. The diagnostic sensitivity of the RAPID-BAP assay, real-time RT-PCR, and conventional RT-PCR in the detection of 34 clinical blood samples suspected to have BEFV infections were 72.73, 36.36, and 18.18%, respectively. The results indicated that the RAPID-BAP assay developed in this study was more sensitive than the conventional RT-PCR and real-time RT-PCR assays for the detection of BEFV. The novel RAPID-BAP assay is an excellent diagnostic tool with high sensitivity, specificity, and fast turnaround time. (c) 2005 Elsevier B.V. All tights reserved.