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

The molecular epidemiological study of foot-and-mouth disease virus (FMDV) has been carried out from different outbreaks in Assam the present study is based on the nucleotide sequencingof circulating FMDV serotype. The samples were subjected to sandwich ELISA, multiplex-PCR and molecular phylogeny to identify the type species. The phylogenetic analysis of virus sequence revealed similarity with theBangladesh isolates in the major branching pattern. The serotype ‘O’has found to be dominant and responsible for most of the recentoutbreaks.Thepersistence of serotype ‘O’ and cytokines expression of IL-1á, IL-1â, IFN-á, TNF-á in blood of recovered animals were done by Real time PCR. The findings indicated that IL-1á, IFN-á and TNF-á genes were up-regulated upto 3 months post infection but IL-1â found to be down regulated with progression of recovery. The present study thus supports that real-time PCR is a powerful technique for reliable detection of persistent FMDV in recovered animals.

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

Journal of Virological Methods ◽

10.1016/j.jviromet.2016.08.002 ◽

2016 ◽

Vol 237 ◽

pp. 114-120 ◽

Cited By ~ 13

Author(s):

Katarzyna Bachanek-Bankowska ◽

Herieth R. Mero ◽

Jemma Wadsworth ◽

Valerie Mioulet ◽

Raphael Sallu ◽

...

Keyword(s):

Real Time ◽

East Africa ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Rt Pcr ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Pcr Assays

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Combining Multiple Assays Improves Detection and Serotyping of Foot-and-Mouth Disease Virus. A Practical Example with Field Samples from East Africa

Viruses ◽

10.3390/v13081583 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1583

Author(s):

Efrem Foglia ◽

Tiziana Lembo ◽

Rudovick Kazwala ◽

Divine Ekwem ◽

Gabriel Shirima ◽

...

Keyword(s):

Real Time ◽

East Africa ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Rt Pcr ◽

Mouth Disease Virus ◽

Field Samples ◽

Foot And Mouth ◽

Pcr Targeting

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012–2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVβ6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.

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GoPrime: Development of an In Silico Framework to Predict the Performance of Real-Time PCR Primers and Probes Using Foot-and-Mouth Disease Virus as a Model

Pathogens ◽

10.3390/pathogens9040303 ◽

2020 ◽

Vol 9 (4) ◽

pp. 303

Author(s):

Emma L A Howson ◽

Richard J Orton ◽

Valerie Mioulet ◽

Tiziana Lembo ◽

Donald P King ◽

...

Keyword(s):

Real Time ◽

Disease Virus ◽

Real Time Pcr ◽

In Silico ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Sequence Variability ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

The Impact

Real-time PCR (rPCR) is a widely accepted diagnostic tool for the detection and quantification of nucleic acid targets. In order for these assays to achieve high sensitivity and specificity, primer and probe-template complementarity is essential; however, mismatches are often unavoidable and can result in false-negative results and errors in quantifying target sequences. Primer and probe sequences therefore require continual evaluation to ensure they remain fit for purpose. This paper describes the development of a linear model and associated computational tool (GoPrime) designed to predict the performance of rPCR primers and probes across multiple sequence data. Empirical data were generated using DNA oligonucleotides (n = 90) that systematically introduced variation in the primer and probe target regions of a diagnostic assay routinely used to detect foot-and-mouth disease virus (FMDV); an animal virus that exhibits a high degree of sequence variability. These assays revealed consistent impacts of patterns of substitutions in primer and probe-sites on rPCR cycle threshold (CT) and limit of detection (LOD). These data were used to populate GoPrime, which was subsequently used to predict rPCR results for DNA templates (n = 7) representing the natural sequence variability within FMDV. GoPrime was also applicable to other areas of the FMDV genome, with predictions for the likely targets of a FMDV-typing assay consistent with published experimental data. Although further work is required to improve these tools, including assessing the impact of primer-template mismatches in the reverse transcription step and the broader impact of mismatches for other assays, these data support the use of mathematical models for rapidly predicting the performance of rPCR primers and probes in silico.

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