DEVELOPMENT AND EVALUATION OF A MULTIPLEX CONVENTIONAL RT-PCR FOR SIMULTANEOUS DETECTION AND TYPING OF FMDV IN WEST AFRICA
Résumé
Introduction:
The West African territories are considered as regions with continuous FMDV circulation where outbreaks of FMDV serotypes O, A, SAT1 and SAT2 have been reported. An early diagnosis of FMD is crucial to implement adequate outbreak management. This study describes the development of a multiplex conventional RT-PCR for both detection and typing of FMD virus circulating in this region, and its evaluation on panels of field samples.
Materials-methods:
The RT-PCR reactions were developed by using primer sets targeting the 3D coding region, the VP1 coding region (O/A/SAT1/SAT2-specific) and the -actin gene in order to produce amplicons of different sizes, easily distinguishable on agarose gel electrophoresis. Two FMDV strains of each targeted serotype as well as two negative samples were used to evaluate intermediate and final RT-PCR protocols. A 6-plex prototype (O/A/SAT1/SAT2/3D/-actin) was finally developed and additionally tested with a panel of reference strains including all serotypes of FMDV. The sensitivity of RT-PCR was evaluated on 24 negative field samples and 37 positive field samples from Benin together with the corresponding virus isolates. This test is currently evaluated on a larger panel of field samples collected in Nigeria and Senegal.
Results:
The 6-plex prototype detected all FMDV strains tested and identified the four serotypes of interest (O/A/SA1/SAT2) without any improper amplification. Using this multiplex protocol, 37 samples from Benin were positive for the 3D target and were correctly serotyped by 6-plex (33) or by 3-plex and simplex RT-PCR (4). 39/40 isolates from Nigeria were properly serotyped using 6-plex (30) or simplex (9). The corresponding field samples as well 39 clinical positive samples from Senegal are under investigation.
Discussion:
We have developed and evaluated a 6-plex RT-PCR that could be easily implemented in diagnostic laboratories in endemic countries, providing thus an improvement for rapid detection and typing of FMDV strains.
Domaines
Sciences du Vivant [q-bio]Origine | Fichiers produits par l'(les) auteur(s) |
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