Improved PCR diagnostics using up-to-date in silico validation: An F-gene RT-qPCR assay for the detection of all four lineages of peste des petits ruminants virus

John Flannery,Paulina Rajko-Nenow,Hannah Arnold,Erik Van Weezep,Piet A Van Rijn,Chanasa Ngeleja,Carrie Batten

doi:10.1016/j.jviromet.2019.113735

John Flannery, Paulina Rajko-Nenow + Show 5 more

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https://doi.org/10.1016/j.jviromet.2019.113735

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Abstract

Peste des petits ruminants (PPR) is a globally significant disease of small ruminants caused by the peste des petits ruminants virus (PPRV) that is considered for eradication by 2030 by the United Nations Food and Agriculture Organisation (FAO). Critical to the eradication of PPR are accurate diagnostic assays. RT-qPCR assays targeting the nucleocapsid gene of PPRV have been successfully used for the diagnosis of PPR. We describe the development of an RT-qPCR assay targeting an alternative region (the fusion (F) gene) based on the most up-to-date PPRV sequence data. In silico analysis of the F-gene RT-qPCR assay performed using PCRv software indicated 98% sensitivity and 100% specificity against all PPRV sequences published in Genbank. The assay indicated the greatest in silico sensitivity in comparison to other previously published and recommended PPRV RT-qPCR assays. We evaluated the assay using strains representative of all 4 lineages in addition to samples obtained from naturally and experimentally-infected animals. The F-gene RT-qPCR assay showed 100% diagnostic specificity and demonstrated a limit of detection of 10 PPRV genome copies per μl. This RT-qPCR assay can be used in isolation or in conjunction with other assays for confirmation of PPR and should support the global efforts for eradication.

Highlights

Peste des Petits Ruminants (PPR) is a viral disease of small ruminants which is a serious threat to food security in countries reliant on small-scale agriculture such as those across the developing world (Banyard et al, 2010)
As PPR has continued to spread since the assays specified in the Organisation for Animal Health (OIE) terrestrial manual were published, we aimed to assess the suitability of these assays in comparison with newly-designed peste des petits ruminants virus (PPRV) RT-qPCR assays
GenBank data files for all PPRV virus isolates (n = 1445) were downloaded from the NCBI taxonomy database and from this, a conservation plot was generated for 99 sequences that were in the area of the genome used for each RT-qPCR assay

Summary

Introduction

Peste des Petits Ruminants (PPR) is a viral disease of small ruminants which is a serious threat to food security in countries reliant on small-scale agriculture such as those across the developing world (Banyard et al, 2010). The global burden of this disease (estimated up to $2.1 USD annually) is well recognised (Baron et al, 2011), PPR is considered for eradication by 2030 by the United Nations Food and Agriculture Organisation (FAO) and the World Organisation for Animal Health (OIE) (FAO, 2015). It is critical that clinical diagnosis is supported by laboratory diagnosis, of which molecular assays present the most sensitive technique. These assays are used in most diagnostic laboratories and a number of conventional and real-time RT-PCR assays are cited in the OIE (World Organisation for Animal Health) terrestrial manual (Libeau and Baron, 2013)

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