Development and application of a reverse transcription droplet digital PCR assay for detection and quantification of Plantago asiatica mosaic virus

Abstract

Plantago asiatica mosaic virus (PlAMV), which infects lilies worldwide, constitutes a potential risk factor for lily production. Development of effective and sensitive methods for the detection and specific quantification of PlAMV is required to prevent the spread of PlAMV in lilies. In this study, we established a sensitive and accurate quantification method for PlAMV infection in lily samples using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity and sensitivity of the proposed RT-ddPCR assay was evaluated for precise detection and quantification of PlAMV. The assay exhibited high specificity for PlAMV and showed no cross-reactivity with other lily viruses. It was about 10-fold more sensitive than reverse-transcription followed by quantitative polymerase chain reaction (RT-qPCR). RT-ddPCR (R2 = 0.9985) and RT-qPCR (R2 = 0.9869) showed high degrees of linearity. In addition, field validation tests on 92 lily samples confirmed that RT-ddPCR (45/92) has higher sensitivity than RT-qPCR (8/92). In summary, RT-ddPCR outperforms RT-qPCR in the analysis of low-viral-load samples and can be utilized for PlAMV infection diagnosis, especially in plant quarantine inspection and PlAMV-free certification programs.