Department of Animal, Plant and Soil Science and Centre for AgriBioscience (AgriBio), La Trobe University, Bundoora, Melbourne, Victoria, Australia.
Department of Economic Development, Jobs, Transport and Resources Centre for AgriBioscience (AgriBio), Victorian Government, Bundoora, Melbourne, Victoria, Australia.
PLoS One. 2018 Sep 27;13(9):e0204310. doi: 10.1371/journal.pone.0204310. eCollection 2018.
Dichelobacter nododus is the causative agent of footrot, a major disease of sheep that creates welfare concerns and large economic loss. The virulence of D. nododus depends on the presence of extracellular proteases, AprV2 and AprB2, which differ by one amino acid. Strains possessing AprV2 can cause clinically virulent disease, while AprB2 may cause clinically benign disease. Current methods for detecting D. nodosus are difficult, laborious and time consuming. New techniques capable of rapidly detecting and typing D. nodosus are needed to aid control programs. Molecular methods, like real-time polymerase chain reaction (rtPCR) can detect aprV2 and aprB2, however, this assay is not field-deployable and cannot support local decision-making during an outbreak. Here we present a field-based molecular assay for detecting aprV2, using loop mediated isothermal amplification (LAMP). The aprV2 LAMP (VDN LAMP) assay was optimised to reliably detect aprV2 from laboratory purified genomic (gDNA) of virulent D. nodosus down to 5x10(-3) ng μL-1, with time to positive (Tp) ≤ 16 minutes, while aprB2 was unreliably detected at 5 ng μL-1 from 16-20 minutes. The use of field collected samples that were rtPCR positive for aprB2 resulted in no amplification, while aprV2 positive field samples by VDN LAMP assay are defined as having Tps' of < 20 minutes and melting temperature between 88.0-88.9°C. When compared to rtPCR, the VDN LAMP was shown to have a diagnostic specificity of 100% and sensitivity of 83.33%. As proof of concept, the VDN LAMP was taken on farm, with all processing occurring in-field. The on farm VDN LAMP successfully detected 91.67% aprV2 positive samples, no aprB2 positive samples (n = 9) or D. nodosus negative (n = 23) samples, with a kappa agreement of 'almost perfect' to rtPCR. This highlights the potential of the assay to inform local treatment decisions for management.
多形拟杆菌是蹄疫的病原体，蹄疫是一种主要的绵羊疾病，会引起福利问题和巨大的经济损失。多形拟杆菌的毒力取决于存在细胞外蛋白酶 AprV2 和 AprB2，它们仅相差一个氨基酸。携带 AprV2 的菌株可引起临床毒力疾病，而 AprB2 可能引起临床良性疾病。目前检测 D. nodosus 的方法既困难又繁琐，耗时较长。需要新的快速检测和分型 D. nodosus 的技术来辅助控制计划。分子方法，如实时聚合酶链反应 (rtPCR)，可以检测 aprV2 和 aprB2，但是，该检测方法无法在现场部署，并且在爆发期间无法支持现场决策。在这里，我们提出了一种基于现场的检测 aprV2 的分子检测方法，即环介导等温扩增 (LAMP)。优化了 aprV2 的 LAMP (VDN LAMP) 检测方法，可从实验室纯化的毒力多形拟杆菌基因组 (gDNA) 中可靠检测到 5x10(-3) ng μL-1 的 aprV2，Tp ≤ 16 分钟，而 aprB2 则在 16-20 分钟时无法可靠检测到 5 ng μL-1。使用 rtPCR 检测为 aprB2 阳性的田间采集样本无扩增，而 VDN LAMP 检测的 aprV2 阳性田间样本的 Tp'定义为<20 分钟，熔点在 88.0-88.9°C 之间。与 rtPCR 相比，VDN LAMP 的诊断特异性为 100%，灵敏度为 83.33%。作为概念验证，该 VDN LAMP 在农场现场进行了检测，所有处理都在现场进行。现场 VDN LAMP 成功检测到 91.67% aprV2 阳性样本，没有 aprB2 阳性样本 (n = 9) 或 D. nodosus 阴性样本 (n = 23)，与 rtPCR 的kappa 一致性为“几乎完美”。这突出了该检测方法为管理提供当地治疗决策信息的潜力。
