The development and deployment of a field-based loop mediated isothermal amplification assay for virulent Dichelobacter nodosus detection on Australian sheep.
The development and deployment of a field-based loop mediated isothermal amplification assay for virulent Dichelobacter nodosus detection on Australian sheep.
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DOI：
10.1371/journal.pone.0204310
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Beddoe T
中科院分区：
文献类型：
--
作者：
Best N;Rodoni B;Rawlin G;Beddoe T
节瘤叉蹄病是一种主要的绵羊疾病，会造成福利问题和巨大的经济损失。毒力测定结果表明，D. nododus依赖于胞外蛋白酶AprV 2和AprB 2的存在，它们相差一个氨基酸。具有AprV 2的菌株可引起临床毒性疾病，而AprB 2可引起临床良性疾病。目前检测D.结节是困难、费力且耗时的。新技术能够快速检测和分型D。需要nodosus来帮助控制程序。分子方法，如实时聚合酶链反应（rtPCR）可以检测aprV 2和aprB 2，但是，这种检测方法不能现场部署，不能支持疫情期间的当地决策。在这里，我们提出了一个基于现场的分子检测aprV 2，使用环介导等温扩增（LAMP）。优化了aprV 2 LAMP（VDN LAMP）测定以可靠地检测来自强毒D.在5 ng μ L-1时，从16-20分钟检测到aprB 2不可靠。使用对aprB 2为rtPCR阳性的田间收集的样品导致无扩增，而通过VDN LAMP测定的aprV 2阳性田间样品被定义为具有< 20分钟的Tps'和88.0-88.9°C之间的解链温度。与rtPCR相比，VDN LAMP的诊断特异性为100%，敏感性为83.33%。作为概念验证，VDN LAMP是在农场进行的，所有处理都在现场进行。现场VDNLAMP检测aprV 2阳性率为91.67%，无aprB 2阳性（n = 9），D. nodosus阴性（n = 23）样品，与rtPCR具有“几乎完美”的kappa一致性。这突出了该测定为管理提供当地治疗决策信息的潜力。
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.
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