The genus capripoxvirus (CaPV), family Poxviridae, includes three virus species: goatpox virus (GPV), sheeppox virus (SPV) and lumpy skin disease virus (LSDV). CaPV causes disease outbreaks with consequent economic losses in Africa and the Middle East. LSDV has recently spread to Southeast Europe. As CaPVs share 96–97% genetic similarity along the length of the entire genome and are difficult to distinguish using serological assays, simple, reliable and fast methods for diagnosis and species differentiation are crucial in cases of disease outbreak. The present study aimed to develop a field-applicable CaPV differentiation method. Nanopore technology was used for whole genome sequencing. A local database of complete CaPV genomes and partial sequences of three genes (RPO30, P32 and GPCR) was established for offline Basic Local Alignment Search Tool (BLAST). Specificities of 98.04% in whole genome and 97.86% in RPO30 gene runs were obtained among the three virus species, while other databases were less specific. The total run time was shortened to approximately 2 h. Functionality of the developed procedure was proved by samples with high host background sequences. Reliable differentiation options for the quality and capacity of hardware, and sample quality of suspected cases, were derived from these findings. The whole workflow can be performed rapidly with a mobile suitcase laboratory and mini-computer, allowing application at the point-of-need with limited resource settings.

The complexity of the current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method (easy express extraction, called TripleE) as a centrifugation-free and electricity-free nucleic acid isolation method. The procedure is based on the well-established magnetic-bead extraction technology using an in-house self-made magnetic 8-channel and a rod cover. With this extraction system, nucleic acids can be isolated with two simple and universal protocols. One method was designed for the extraction of the nucleic acid in resource-limited “easy labs”, and the other method can be used for RNA/DNA extraction in the field for so-called molecular “pen-side tests”. In both scenarios, users can extract up to 8 samples in 6 to 10 min, without the need for any electricity, centrifuges or robotic systems. In order to evaluate and compare both methods, clinical samples from various viruses (African swine fever virus; lumpy skin disease virus; peste des petits ruminants virus; bluetongue virus), matrices and animals were tested and compared with standard magnetic-bead nucleic acid extraction technology based on the KingFisher platform. Hence, validation data were generated by evaluating two DNA viruses as well as one single-stranded and one double-stranded RNA virus. The results showed that the fast, easy, portable and electricity-free extraction protocols allowed rapid and reliable nucleic acid extraction for a variety of viruses and most likely also for other pathogens, without a substantial loss of sensitivity compared to standard procedures. The speed and simplicity of the methods make them ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings.