Department of Animal Science, Institute of Animal Science, Biotechnology and Nature Conservation, Faculty of Agricultural and Food Sciences and Environmental Management, Böszörményi ut. 138, University of Debrecen, Debrecen 4032, Hungary - Doctoral School of Animal Science, University of Debrecen, Debrecen 4032, Hungary.
Department of Animal Science, Institute for Agricultural Research and Educational Farm, Faculty of Agricultural and Food Sciences and Environmental Management, Böszörményi ut. 138, University of Debrecen, Debrecen 4032, Hungary - Doctoral School of Animal Science, University of Debrecen, Debrecen 4032, Hungary.
Parasite. 2021;28:82. doi: 10.1051/parasite/2021078. Epub 2021 Dec 15.
In this study, we present an optimised colourimetric and a lateral flow LAMP assay for the detection of Haemonchus contortus in small ruminant faecal samples. Using a previously published LAMP primer set, we made use of commercially available colourimetric LAMP and lateral flow kits and combined this into an optimised diagnostic assay which was then tested on field faecal samples from Eastern and South-Eastern Hungary as well as a pure H. contortus egg faecal sample from Košice, Slovakia. Both assays showed no conflicts in visual detection of the results. Additionally, we modified and tested several centrifuge-free DNA extraction methods and one bead-beating egg lysis DNA extraction method to develop a true point of care protocol, as the source of the starting DNA is the main rate-limiting step in farm-level molecular diagnosis. Out of the various methods trialed, promising results were obtained with the magnetic bead extraction method. Sample solutions from the Fill-FLOTAC technique were also utilised, which demonstrated that it could be efficiently adapted for field-level egg concentration to extract DNA. This proof of concept study showed that isothermal amplification technologies with a colourimetric detection or when combined with a lateral flow assay could be an important step for a true point of care molecular diagnostic assay for H. contortus.
在本研究中，我们提出了一种优化的比色法和侧向流动环介导等温扩增（LAMP）检测方法，用于检测小反刍动物粪便样本中的捻转血矛线虫。我们使用先前发表的 LAMP 引物组，利用市售的比色 LAMP 和侧向流动试剂盒，并将其组合成一种优化的诊断检测方法，然后在匈牙利东部和东南部的现场粪便样本以及来自斯洛伐克科希策的纯捻转血矛线虫卵粪便样本上进行测试。两种检测方法在视觉检测结果方面均无冲突。此外，我们还修改并测试了几种无离心 DNA 提取方法和一种珠磨卵裂解 DNA 提取方法，以开发真正的即时检测协议，因为起始 DNA 的来源是农场级分子诊断的主要限速步骤。在试用的各种方法中，磁性珠提取方法获得了有希望的结果。还利用了 Fill-FLOTAC 技术的样品溶液，表明它可以有效地适用于现场级别的卵浓缩以提取 DNA。这项概念验证研究表明，具有比色检测的等温扩增技术或与侧向流动检测相结合，可能是捻转血矛线虫真正的即时检测分子诊断检测方法的重要步骤。
