During the last 25 years, swine vesicular disease (SVD) has occurred in Italy mostly sub-clinically. Therefore, regular testing of fecal samples from suspected holdings and high turnover premises was fundamental to identifying virus circulation and to achieve SVD eradication. In this study, we evaluated diagnostic performances of six genomic amplification methods, using positive fecal samples from 78 different outbreaks (1997–2014), which included different lineages. Comparison of three RT-PCRs, designed to amplify the same 154 nt portion of the gene 3D, demonstrated that a conventional and a real-time based on SYBR Green detection assay showed the highest diagnostic sensitivity, detecting all samples, while a real-time TaqMan-based test missed three cases, owing to two mismatches in the probe target sequence. Diagnostic and analytical specificities were optimal, as 300 negative field samples and other enteroviruses reacted negative. Three further evaluated tests, previously described, were a 3D-targeted reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and two real-time RT-PCRs targeted on the 5′UTR region. Here, the presence of multiple mismatches in probe and primers reduced the diagnostic performances, and two of the assays were unable to detect viruses from one sub-lineage. These results highlight that the choice of tests using less nucleotide targets significantly contributed to the success of the SVD eradication plan.

中文翻译：

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在意大利流行的基因组谱系中，检测不同基因组扩增方法检测猪水泡病病毒的诊断性能

在过去的25年中，意大利的猪水疱病（SVD）大多是亚临床的。因此，对可疑馆藏和高周转场所的粪便样本进行定期测试对于确定病毒传播和根除SVD至关重要。在这项研究中，我们使用来自78种不同爆发（1997-2014年）的阳性粪便样本（包括不同谱系）评估了六种基因组扩增方法的诊断性能。设计用于扩增基因3D的154 nt部分的三个RT-PCR的比较表明，常规和基于SYBR Green检测的实时检测方法显示出最高的诊断灵敏度，可检测所有样品，而实时检测基于TaqMan的测试由于探针目标序列中的两个错配而错过了三例。诊断和分析的特异性最佳，因为300个阴性野外样品和其他肠道病毒反应呈阴性。先前描述的三个进一步评估的测试是3D靶向逆转录酶环介导的等温扩增（RT-LAMP）和两个针对5'UTR区域的实时RT-PCR。在此，探针和引物中存在多个错配会降低诊断性能，并且其中两种测定法无法从一种亚谱系中检测出病毒。这些结果表明，使用较少核苷酸靶标进行测试的选择显着促进了SVD根除计划的成功。分别是靶向3D的逆转录酶环介导的等温扩增（RT-LAMP）和靶向5'UTR区域的两个实时RT-PCR。在此，探针和引物中存在多个错配会降低诊断性能，并且其中两种测定法无法从一种亚谱系中检测出病毒。这些结果表明，使用较少核苷酸靶标进行测试的选择显着促进了SVD根除计划的成功。分别是靶向3D的逆转录酶环介导的等温扩增（RT-LAMP）和靶向5'UTR区域的两个实时RT-PCR。在此，探针和引物中存在多个错配会降低诊断性能，并且其中两种测定法无法从一种亚谱系中检测出病毒。这些结果表明，使用较少核苷酸靶标进行测试的选择显着促进了SVD根除计划的成功。