Development of a Multiplex RT-PCR for Simultaneous Detection of Five Actinidia Viruses

Abstract

Kiwifruit (Actinidia spp.) is a perennial fruit tree, and the fruit of kiwifruit is economically and nutritionally important worldwide. To date, approximately 23 species of kiwifruit viruses have been reported worldwide. As for the detection method for kiwifruit viruses, previous reports mostly used the single RT-PCR detection method. In the detection of kiwifruit viruses, multiplex RT-PCR has the advantages of being fast, reliable and inexpensive. In this study, a stable, efficient and reliable multiplex RT-PCR method for the detection of the five most common kiwifruit viruses was established. The concentrations of Mg2+ and HS-Taq and the annealing temperature in the multiplex PCR system were optimized. The results indicate that the optimal annealing temperature was 56 °C; the optimal concentration of added Mg2+ was 2 mM; and the optimal concentration of HS-Taq was 1.0 U/μL. The stability of the optimized multiplex RT-PCR system was verified by field sample testing, and the results showed that the multiplex RT-PCR system was stable, efficient and reliable. This will provide much convenience for the detection of kiwifruit viruses in the future.