Standardization of RT-PCR protocol for detection and typing of emerging food and mouth disease virus in lebanon

Abstract

The applicability of developed RT-PCR protocols at OIE/FAO World Reference Laboratory in diagnostic of FMD is challenged by the continuous emergence of new lineage of isolates around the world, and specifically in the Eastern Mediterranean region where the genetic studies are very limited. The purpose of RT-PCR standardization is to amplify the conserved 5’ UTR gene of FMD virus, using an application of variable RNA concentrations and cycling conditions on two tongue-epithelial tissue samples, collected from cows (FMD1 and FMD2) provided by the Lebanese Ministry of Agriculture, to optimize the FMD typing, and to apply the established protocols on other suspected cases of FMD. The RNA amount was set in a volume of 4μL at 20 ng/μL and the optimal RT-PCR cycling condition was established at 50°C for 45 minutes, followed by 15 minutes at 95°C, then by 30 cycles of 94°C for 1 minute, a lower temperature of 55°C for 1 minute, then a higher temperature of 72°C for 2 minutes, followed by one cycle at 72°C for 7 minutes. The optimized amplification resulted in 328 bp band from the two field samples (FMD1 and FMD2). The optimized RT-PCR protocol applied on four additional field samples collected from two lactating cows, revealing two additional positive isolates of FMD (FMD3 and FMD4). The RT-PCR was used in typing of the positive FMD samples with different sets of primers specific for various FMD types, revealing SAT2 type in samples FMD1, FMD3 and FMD4, and failed in typing the FMD2 sample. The importance of the optimization of RT-PCR, in uncovering the presence of new types of FMD in livestock of this region, and in establishment of future FMD control programs, is discussed. © 2014, Veterinary Solutions LLC. All rights reserved.