Abstract
Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals that causes severe economic losses. The disease is characterized by a vesicular condition and it cannot be differentiated from other vesicular diseases. Therefore, laboratory confirmation of any suspected FMD case is compulsory. Despite viral isolation in cell cultures has been considered for many years as the gold standard for FMD diagnosis, the advantages of real-time reverse transcription polymerase chain reaction (rRT-PCR) technology have motivated its use directly in clinical specimens for FMD diagnosis. The current work was aimed to develop and validate a molecular multi-check strategy using rRT-PCR (mMulti-rRT-PCR) based on SYBR-Green I for pan/foot-and-mouth disease virus (pan/FMDV) diagnosis. From in silico approaches, different primer pairs previously reported were selected and modified to reduce the likelihood of viral escape as well as potential failures in the pan/FMDV detection. The analytical parameters were evaluated using a high number of representative viral strains. The repeatability of the assay and its performance on field samples were also assessed. The mMulti-rRT-PCR was able to detect emergent FMDV strains that circulated in South America between the years 2006–2010 and on which the single rRT-PCRs failed when they were applied independently. The results obtained here showed that the proposed system is an accurate and rapid diagnosis method for sensitive and specific detection of FMDV. Thus, a validated mMulti-rRT-PCR assay based on SYBR-Green I detection coupled to melting curves resolution for pan/FMDV diagnosis on clinical samples is proposed. This study also highlights the need to incorporate the multi-target detection principle in the diagnosis of highly variable agents, specially, of those listed by OIE like FMDV.
Highlights
Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals including cattle, pigs, sheep, goats and around 70 wildlife species [1]
Cell Culture and Virus Propagation Viruses were grown and titrated in PK15 cell line (ATCC R CCL-33TM) (CSFV strains), MDBK cell line (ATCC R CCL22TM) (BVDV and border disease virus genotype 1 (BDV) strains) and BHK-21 (ATCC R CCL10TM) (FMDV and vesicular stomatitis virus (VSV) strains/isolates) following standard procedures described in the Organization for Animal Health (OIE) Manual [26,27,28,29,30]
Inspecting the entropy values for the last four nucleotides at the 3 ′OH of the primers, it was observed that, excepting the primer pair FP/RP-TgR3, the rest showed three nucleotides with values greater than 0.1, proving they are highly susceptible to priming failure (Figure 1)
Summary
Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals including cattle, pigs, sheep, goats and around 70 wildlife species [1]. Despite the high morbidity of the disease, mortality is low and mostly restricted to young animals [2]. FMD causes severe economic losses due to both significant decrease in production efficiency of the animals affected and sanitary control measures costs [3]. The viral genome is about 8,400 nt in length and consists of a single open reading frame (ORF) flanked by two untranslated regions (UTRs). The virus replicates extremely rapid with a high mutation rate, generating viral populations with genetically diverse genomes known as viral quasispecies [4]
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