Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing

A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green 1, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures (T(m)s); 84.3-84.43 degreesC for D isolates, and 85.34-86.11 degreesC for M isolates. The associated amplicon sizes were 114 and 380 bp, respectively. The procedure was used for detection and identification of PPV in both herbaceous and woody hosts. The T-m for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Universal primers included in the study detected all isolates of PPV tested, amplifying a 74 bp fragment. The T-m of this fragment varied from 80.12 to 81.52 degreesC and may have supplementary value for PPV identification. SYBR Green-based detection was compared to detection using a hybridization LUX fluorogenic primer. Better resolution of the melting peaks was observed with SYBR Green 1, than with the LUX primers, hence strain identification with SYBR Green I was more reliable. This is a simple approach to PPV strain identification with the relatively inexpensive dye SYBR Green 1, and eliminates any need for electrophoretic analysis of amplicons or RFLP patterns using ethidium bromide. Crown Copyright (C) 2004 Published by Elsevier B.V. All rights reserved.