SPOT REAL-TIME RT-PCR: A METHOD FOR DIRECT DETECTION OF PLUM POX VIRUS AVOIDING RNA EXTRACTION

Spot real-time RT-PCR is a method for detection of Plum pox virus using conventional ELISA plant crude extracts immobilised on paper without the need of RNA extraction. The method was validated on different prunus field samples collected in winter and springtime. Seventy-nine percent of accuracy was obtained on winter samples and 97% in samples collected in spring. The sensitivity of spot real-time RT-PCR technique was compared with those obtained by DASI-ELISA, Co-PCR and conventional real-time RT-PCR using RNA as template. Spot real-time RT-PCR resulted 100-times more sensitive than DASI-ELISA and 10-times more sensitive than Co-PCR, although conventional real-time PCR was the most sensitive method for PPV detection. Joined to these advantages, spot real-time RT-PCR permits the storage of immobilised samples on paper at room temperature or frozen for long periods without decreasing the sensitivity of detection.