Detection Of Peach Viruses By Multiplex RT-PCR

In China, Peach trees are widely cultivated, but in recent years, the infection of peach viruses was severe. Peach viruses affect the normal development of peach fruit trees’ industry. There are4major peach viruses that have been reported:ACLSV (Apple chlorotic leaf spot virus); PNRSV (Prunus necrotic ring spot virus); CGRMV (Cherry green ring mottle virus); APCLSV (Apricot pseudo-chlorotic leaf spot virus), and they were also infected with other viruses. From2011to2012,505peach samples were randomly collected from twelve provinces where peach trees are grown. The samples were tested by RT-PCR and double-antibody sandwich ELISA (DAS-ELISA). The overall average of virus infection level in peach trees was24.6%.In this study, we established a multiplex PCR system to detect ACLSV, CGRMV, PNRSV and APCLSV simultaneously on the basis of single RT-PCR. We designed four specific primer pairs according to conserved regions of respective viruses, and optimized the reaction components. We detected ACLSV, CGRMV, PNRSV and APCLSV in mixed infection peach samples successfully and obtained four bands consistent with the experimental design:ACLSV (632bp), CGRMV (439bp), PNRSV (346bp) and APCLSV (282bp). Sequence homology analysis confirmed the reliability of the mRT-PCR system.In addition, a duplex real-time PCR approach for detection of ACLSV and CGRMV was developed in this study. The duplex real-time PCR results were100%consistent with RT-PCR and single real-time PCR for field detection of peach trees. Real time PCR assay was applied to detect ACLSV genomic RNA、CGRMV genomic RNA and RPII mRNA, UBQ10mRNA in different infected peach issues. Single real-time PCR showed that ACLSV and CGRMV in peach accumulate to higher levels in bark than in leaves or flowers, and the ACLSV titer was～1%that of CGRMV. A duplex SYBR Green I-based PCR assay was developed to simultaneously detect these viruses.This is the first multiplex SYBR Green I real-time PCR assay described for plant virus detection.