Department of Jobs, Precincts and Regions, Agriculture Victoria Research, AgriBio, Bundoora, Vic., Australia.
Department of Jobs, Precincts and Regions, Biosecurity and Agriculture Services, Epsom, Vic., Australia.
Transbound Emerg Dis. 2020 Nov;67(6):2494-2506. doi: 10.1111/tbed.13589. Epub 2020 May 6.
Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-for-purpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs).
口蹄疫（FMD）是一种高度传染性的偶蹄动物病毒病。全球暴发凸显了在先前“无口蹄疫”国家发现疾病可能带来的重大经济、贸易、心理社会和动物福利影响。快速和早期诊断对口蹄疫的控制和减轻有害后果具有重要意义。我们描述了进一步开发和验证使用已发表的 LAMP 引物套件的基于逆转录环介导等温扩增的口蹄疫病毒（RT-LAMP-FMDV）测试的过程，该测试可用于现场。设计并引入了内部阳性对照（IPC）以与该测定一起使用，以减轻未提取的现场样本的任何固有干扰并避免假阴性。还包括进一步的修改以提高测试的速度和可操作性，以便在现场条件下由未经实验室培训的人员使用，使用具有稳定货架期的反应试剂盒，这些试剂盒需要最低限度的液体处理技能。我们研究了该测定与针对口蹄疫病毒 3D 区的基于实验室的实时逆转录聚合酶链反应（rRT-PCR）测试（Tetracore Inc）的性能比较。LAMP 有可能作为当前实验室诊断（如 rRT-PCR）的补充，作为口蹄疫的初步诊断工具。我们描述了一种使用来自泰国的各种血清型的提取 RNA 样本在现场验证该测试的策略方法，然后最终使用来自不丹和澳大利亚的疑似口蹄疫动物（主要是口腔病变拭子）的未提取现场样本进行验证。验证的统计方法是使用频率主义和贝叶斯潜在类别方法进行的，这两种方法都证实了这种新的 RT-LAMP-FMDV 测试适用于作为 herd 诊断工具，具有>99%的诊断特异性和 79%的敏感性（95%贝叶斯可信区间：65，90%）在未提取的现场样本（口腔拭子）上。
