Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.
J Virol Methods. 2011 Mar;172(1-2):8-15. doi: 10.1016/j.jviromet.2010.12.003. Epub 2010 Dec 15.
African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 10⁹ to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10⁻¹ to 10⁻⁵. The detection limit was 10⁻⁵ dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field.
非洲猪瘟病毒（ASFV）是一种高致病性的 DNA 病毒，是非洲猪瘟（ASF）的病原体，这种传染病可感染所有品种和年龄段的家猪和野猪，引起多种综合征。急性疾病的特征是高热、网状内皮系统出血和高死亡率。一种基于线性聚合酶链反应（LATE-PCR）原理的强大新型诊断检测方法被开发出来用于检测 ASFV。LATE-PCR 是一种不对称 PCR 的高级形式，可直接扩增大量的单链 DNA。在 PCR 扩增后，通过终点分析获取荧光读数。通过熔解曲线分析验证扩增产物的正确性。该检测方法设计用于扩增 ASFV 基因组的 VP72 基因。对 19 株 ASFV 细胞培养病毒株和来自感染实验猪的 3 个组织样本（脾脏、扁桃体和肝脏）进行了检测。所有细胞培养和组织样本中均检测到了病毒。在检测的 5 种 ASFV 相关病毒中，没有一种产生阳性信号，表明该检测方法具有高度特异性。在两个单独的实时单重反应中，使用合成 ASFV 和合成对照 DNA 靶标进行了 LATE-PCR 检测的灵敏度测定，这些靶标从 10⁹ 到 1 个初始拷贝/反应连续稀释。检测限分别为 1 和 10 拷贝/反应。在包含 150 个合成对照 DNA 恒定水平和从 10⁻¹ 到 10⁻⁵连续稀释的临床脾脏组织样本的双重终点反应中，也测试了该检测方法的灵敏度。检测限为 10⁻⁵ 稀释度，相当于大约 1 个拷贝/反应。由于该检测方法旨在用于实验室环境或便携式 PCR 仪器（Smiths Detection，英国沃特福德的 Bio-Seeq 便携式兽医诊断实验室）中，因此 LATE-PCR 为实验室和现场的 ASF 诊断提供了一种强大而新颖的工具。
