Development and preliminary application of a reverse-transcription droplet digital PCR assay for detection and quantification of apple stem pitting virus in vitro propagated apple plantlets

Apple stem pitting virus (ASPV) is a viral pathogen that affects pomefruit trees and causes significant economic losses. Considering its global distribution and economic impact, developing accurate and sensitive diagnostic technologies is imperative for producing ASPV-free plantlets through in vitro micropropagation. In the current study, the complete genome sequence of ASPV was obtained through high-throughput sequencing of micro-propagated apple plantlets. Further, the genome sequence was used to design primer sets for detecting ASPV using reverse-transcription droplet digital PCR (RT-ddPCR). The optimized RT-ddPCR assay did not show cross-reactivity to other major apple viruses, and it was at least 10 times more sensitive than RT-real-time quantitative PCR (RT-qPCR). Both RT-ddPCR and RT-qPCR data exhibited high degrees of linearity, with an R2 value of 0.9982. In addition, a field test was performed to validate the RT-ddPCR data in 44 apple plantlets. The results revealed that ddRT-PCR detected 41 positive samples, whereas only 39 positive samples were detected by RT-qPCR. The developed RT-ddPCR assay can serve as a precise, sensitive, and quantifiable method for the reliable diagnosis of ASPV infection in micropropagated apple plantlets, particularly in ASPV-free certification programs. Its utilization contributes to improved disease management, ensuring the maintenance of apple production.