Development of a Multiplex PCR for Simultaneous Detection of Blueberry Red Ringspot Virus and Blueberry Scorch Virus Including an Internal Control

Abstract

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.