"FastCheckFLI PPR-like"-A Molecular Tool for the Fast Genome Detection of PPRV and Differential Diagnostic Pathogens

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 Mycoplasmacapricolumsubsp.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 "FastCheck^FLI 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.

Keywords: Small ruminant morbilli virus; differential diagnosis; fast extraction; high-speed RT-qPCR; molecular pen-side test; peste des petits ruminants virus (PPRV); rapid detection method.

Figure 1

Workflow of the study design illustrating the sole experiments leading to the establishment of “FastCheck^FLI PPR-like”.

Figure 2

Test series for the establishment of a speed-optimized extraction protocol: differences in the Cq-values of the original (“original”) and the maximum shortened protocols (“short 4” and “short 5”) for three different extraction kits: VET kit; CADOR kit and CORE kit using a BioSprint 15 platform.

Figure 3

Test series for the establishment of a high-speed RT-qPCR: differences in the Cq-values of the standard (“standard”) and maximal shortened protocols (“short 4” and “short 5”) for two different primer–probe mixtures: Polci-mix and PPRV-mix 6.

Figure 4

Device test with five qPCR cyclers: differences in Cq-values of the standard and short protocol 5 for the Polci-mix.

Figure 5

Test series with three lyophilized kits and five different pathogens: comparison of a well-established PCR protocol (named “AgPath” or “Qiagen”), the standard protocol (named “standard”) of the lyophilized kit, and the maximal shortened protocol (named “No.5”) of the corresponding kit. The kits used for the validation of the dilution series: AgPath = AgPath-ID™ One-Step RT-PCR Reagents; Qiagen = QuantiTect Multiplex-PCR Kit NO ROX. The three lyophilized kits used: Taykon kit; Qscript kit and Capital kit. The five different pathogens tested: PPRV, FMDV, Parapoxvirus ovis, GTPV, Mccp.