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Improvement of LCHV-1 detection by conventional RT-PCR and Real Time PCR protocols

Nicola Fiore: University of Chile

<div>Sweet cherry is one of the most important fruit crops in economical income in Chile, placing the country in the first place in cherry production in the southern hemisphere. From October 2015 to March 2016, a survey was performed in the main sweet cherry producing regions of Chile, collecting samples from 223 trees. Several viruses were detected by RT-PCR, but not <em>Little cherry virus 1</em> (LChV-1), using primer pairs indicated in literature. Nineteen samples were randomly selected for small RNA sequencing in Illumina MiSeq platform and sample reads were trimmed using FASTX and assembled using VELVET. Four samples presented contigs that matched with LChV-1 Genbank references, indicating the presence of new variants of the virus in Chilean samples. As a consequence and in order to improve the detection of LCHV-1 by conventional RT-PCR and Real time PCR TaqMan, new specific primer pairs and a molecular probe were designed using the 3' untranslated region of the virus genome. Using the total nucleic acid extracts stored at -80˚ C, the same 223 samples were analyzed again, obtaining 15% and 30% of positive samples with conventional RT-PCR and Real time PCR TaqMan, respectively. These new detection tools allowed the improvement of the detection of LChV-1, being Real Time PCR the most sensitive protocol. In addition, this last method was developed for the first time for the detection of LChV-1.</p> <p>Acknowledgements. This work was supported by project FONDEF Idea ID15I10087, CONICYT, Chile.</div>