Vývoj a validace diagnostického real-time PCR systému pro detekci Olive latent viru 1 (OLV-1) v rostlinách jahodníku

Abstract

Olive (Olea europaea L.) is a natural host of Olive latent virus 1 (OLV-1) that was also found in citrus trees, tulips and tomatoes. OLV-1 was detected by next-generation sequencing (NGS) also in strawberry plants, and therefore a new real-time PCR system has been developed for its routine detection. The designed diagnostic kit was validated following the PM 7/98 (4) standard issued by European and Mediterranean Plant Protection Organization (EPPO) to allow its use in accredited laboratories. Using a synthetic standard, the following performance parameters of the kit were set during validation: specificity, sensitivity, repeatability, and reproducibility. Based on available OLV-1 sequences and our own sequencing results, primers and probe were designed to specifically detect only OLV-1 isolates (100% specificity). Sensitivity of the detection was assessed to be 50 copies per reaction; 500 copies per reaction was used to calculate other parameters. Repeatability and reproducibility were expressed as a variation coefficient, and were found to be 1.05 % and 1.08 %, respectively. Obtained parameters proved that the real-time PCR system for the detection of OLV-1 in strawberries was functional, reliable, and could be used in accredited laboratories for valid OLV-1 diagnostic.

Keywords

• molecular methods
• accredited laboratory
• plant viruses
• sensitivity
• specificity
• repeatability