Abstract
To assist the global eradication of peste des petits ruminants virus (PPRV), a molecular test for the rapid and reliable detection of PPRV was developed which additionally enables the detection of pathogens relevant for differential diagnostics. For this purpose, the necessary time frame of a magnetic bead-based nucleic acid extraction protocol was markedly shortened to 7 min and 13 s. The optimized extraction was run on a BioSprint 15 platform. Furthermore, a high-speed multi-well RT-qPCR for the genome detection of PPRV and additional important pathogens such as Foot-and-mouth disease virus, Parapoxvirus ovis, Goatpox virus, and Mycoplasma capricolum subsp. capripneumoniae was established and combined with suitable internal control assays. The here-described qPCR is based on a lyophilized master mix and takes only around 30 to 40 min. Several qPCR cyclers were evaluated regarding their suitability for fast-cycling approaches and for their diagnostic performance in a high-speed RT-qPCR. The final evaluation was conducted on the BioRad CFX96 and also on a portable Liberty16 qPCR cycler. The new molecular test designated as “FastCheckFLI PPR-like”, which is based on rapid nucleic acid extraction and high-speed RT-qPCR, delivered reliable results in less than one hour, allowing its use also in a pen-side scenario.
Highlights
Peste des petits ruminants (PPR) is a viral disease of domestic and wild ruminants with a wide range of susceptible species of the order Artiodactyla [1,2,3,4]
Several qPCR cyclers were evaluated regarding their suitability for fast-cycling approaches and for their diagnostic performance in a high-speed RT-qPCR
The genome of peste des petits ruminants virus (PPRV) consists of 15,948 nucleotides, which encode for six structural (nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), large protein (L)) and two non-structural proteins (C and V) [6]
Summary
Peste des petits ruminants (PPR) is a viral disease of domestic and wild ruminants with a wide range of susceptible species of the order Artiodactyla [1,2,3,4]. Mccp triggers a disease mainly focused on the respiratory tract of goats [19] It is mainly present in countries in Africa, the Middle East, and Asia [20]. RT-RPAs deliver results within 20 min, being less time-consuming for a molecular diagnostic test, and show a good diagnostic performance compared to RT-qPCR These molecular assays have shown drawbacks regarding their detection limit and their reproducibility when compared to RT-qPCRs [25]. The idea behind the “FastCheckFLI PPR-like” was to develop a diagnostic tool that focuses on fast and flexible PPRV diagnostics integrating the detection of additional pathogens, since the clinical similar signs of that pathogens complicate reliable diagnostics in the field. The presented data of our study should support the molecular diagnostics and differential diagnosis of PPR in small ruminants using universal available nucleic acid extraction and PCR applications
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