Detection of three closteroviruses in stone fruit trees by multiplex assays

Simultaneous detection of Plum bark necrosis stem pitting- associated virus (PBNSPaV), Little cherry virus 1 (LChV-1) and Little cherry virus 2 (LChV-2) was achieved with a quadruplex one-step RT-PCR, and a hybridization test with a multi-riboprobe, apparently representing the first successful attempt of concomitant identification of viruses of the family Closteroviridae in stone fruit trees. Quadruplex RT-PCR detected double (natural and artificial) and triple (artificial) infections in all samples tested, amplifying also a plant mRNA as an internal control. Standard and quadruplex RT-PCR were comparable in terms of detection limits and specificity. Molecular hybridization with a multi-riboprobe (‘clotri’) containing partial sequences of PBNSPaV, LChV-1 and LChV-2, allowed the detection of each of the three viruses in Prunus hosts. The sensitivity of single riboprobes and “clotri” was comparable.