Published in Scientific Bulletin. Series F. Biotechnologies, Vol. XX
Written by Maria Rodica GURAU, Stelian BARAITAREANU, Marius Andrei MANESCU, Mihaela Cristiana POPP, Doina DANES
Several molecular methods have been developed for diagnostic or surveillance of those agents of emerging infectious diseases, including for the Schmallenberg-Simbu group viruses. Serological surveillance of the Schmallenberg-Simbu group viruses in Romania revealed the presence of positive ruminants and it rise up the question about the presence of virus into the environment. In this frame, the paper has described preliminary studies concerning the optimisation of classical RT-PCR of pan-Simbu virus group. We used the OneStep RT-PCR Kit and made minor changes as follows. For one reaction were used 5 μl 5x OneStep RT-PCR Buffer, 1.5 μl dNTP 10 mM, 1.5 μl OneStep RT-PCR Enzyme Mix, 4 μl primer panOBV-L-2959 F 10 μM, 4 μl primer panOBV-L-3274R 10 μM and 9 μl RNase-free water. Into reaction tubes were transferred 25 μL master mix + 10 μL sample. Thermal cycling program consisted of one cycle of 50°C - 30 min and one cycle of 95°C - 15 min, followed by 42 cycles of 95°C - 30 s, 55°C - 30 s, 72°C - 30 s and 72°C - 10 min. All results obtained by real time RT-PCR (virotype SBV RT-PCR Kit) and classical RT-PCR were correlated with the quantity of estimated RNA by fluorometry. The sensitivity of classical RT-PCR was lower than sensitivity of real time RT-PCR, the positive result being acquired at a minimum of 3.91 ng/μl RNA per sample. The specificity of methods was the same, without non-specific electrophoretic bands detection. Therefore, our classical RT-PCR protocol can be a useful tool in evaluation of virus circulation in countries with or without history of associated Simbu disease in livestock, or with reported seroconversion.
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