Triplex real-time PCR assay for sensitive and simultaneous detection of grapevine phytoplasmas of the 16SrV and 16SrXII-A groups with an endogenous analytical control

Flavescence doree (FD) and Bois noir (BN) are the two main yellows of grapevine in Europe and are caused by phytoplasmas of the 16SrV and 16SrXII-A groups respectively. A new triplex real-time PCR assay was developed in order to detect simultaneously the FD and BN phytoplasmas as well as grapevine chloroplastic DNA with TaqMan minor groove binder probes. Each set of designed primers and probes specifically detected the map gene of the FD and BN phytoplasmas, respectively and did not detect phytoplasmas from other phylogenetic groups. PCR efficiencies varied from 90 to 110 %. The PCR assay showed good intra-test and inter-test reproducibility. Triplex real-time PCR was compared to the conventional biplex nested-PCR method. The sensitivity of the real-time PCR, tested on several infected periwinkle and grapevine samples, was up to 5 and 100 times higher for the BN-P and the FD-P targets, respectively. Out of 109 grapevine samples analysed 10, which were negative with the nested PCR, turned to be FD-P positive with the real-time PCR. A decision scheme was set up according to the Ct values of the FD-P, BN-P and grapevine targets in order to assess the routine detection results.