Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

Abstract

Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce C values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region.

Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform

“…In these situations, it is beneficial to characterize circulating FMDV outbreaks in order to make tailored control programmes a realistic possibility (Namatovu et al., ). In support of this, this study also evaluated a lyophilized version of a published FMDV‐typing assay specific to East Africa (Bachanek‐Bankowska et al., ). The typing assay was able to characterize FMDV present in four different sample types collected from seven small holdings (three in Kenya, three in Tanzania and one in Ethiopia) where cattle were presenting two to seven‐day‐old FMD lesions.…”

“…Samples considered positive by the lyophilized pan‐serotype‐specific assay ( n = 46), in addition to a selection of samples from cattle considered clinically negative ( n = 7), were then subsequently characterized using the East Africa‐specific typing assay which has been developed to detect all known FMDV strains relevant to this region (Bachanek‐Bankowska et al., ). Of these, 24 were identified as either A or O (Figure ); no amplification was present for samples collected from clinically FMD‐negative cattle.…”

“…The East Africa‐specific typing rRT‐PCR used the same chemistry, with the primers and probes as previously published in Bachanek‐Bankowska et al. (). In the typing assay, probes for each serotype were modified for multiplex detection using the following fluorescence channels: O (Dragonfly Orange™), A (FAM), SAT 1 (Cy ® 5) and SAT 2 (Texas Red ® ).…”

“…This study evaluates the performance of a commercially available lyophilized FMDV pan‐serotype‐specific rRT‐PCR assay (TC‐9092‐064; Tetracore, Inc., MD, USA), and a newly available East Africa‐specific typing assay (Bachanek‐Bankowska et al., ), both performed on the T‐COR™ 8 (Tetracore, Inc.), a portable, battery‐powered thermocycler (Almassian, Cockrell, & Nelson, ). The assessment was performed within laboratory and East African field settings, providing an approach that can type FMDV in situ using molecular methods.…”

Opportunities for enhanced surveillance of foot‐and‐mouth disease in endemic settings using milk samples

“…The OIE recommended pan‐serotypic rRT‐PCR assay was carried out on an Applied Biosystems ® 7500 Real‐time PCR System, using the Superscript III Platinum ® One‐Step qRT‐PCR Kit (Invitrogen ™ ), with primers and probes targeting the conserved three‐dimensional region of the FMDV genome (Callahan et al., ; OIE Terrestrial Manual, ), and thermal cycling conditions as previously reported (Shaw et al., ). Positive samples were then tested using East Africa (EA) typing rRT‐PCR assays, as previously described (Bachanek‐Bankowska et al., ). RNA extracted from cell culture isolates TAN/39/2012, TAN/6/2013, TAN/33/2014 and TAN/19/2012 (Table S1) supplied by the WRLFMD were used as rRT‐PCR assay positive controls.…”