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

• Published in: Crop protection 2023-07, Vol.169, p.106255, Article 106255
• Main Authors: Lee, Hyo-Jeong, Choi, Sena, Cho, In-Sook, Yoon, Ju-Yeon, Jeong, Rae-Dong
• Format: Article
• Language: English
• Subjects: certification; cross reaction; Detection; droplets; plant protection; Plantago asiatica mosaic virus; quantitative polymerase chain reaction; quarantine; reverse transcription; Reverse transcription droplet digital PCR; Reverse transcription quantitative polymerase chain amplification; risk factors
• ISSN: 0261-2194; 1873-6904
• DOI: 10.1016/j.cropro.2023.106255

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. •RT-ddPCR assay was developed for the detection and quantification of Plantago asiatica mosaic virus (PlAMV).•RT-ddPCR assay exhibited high sensitivity and specificity for detecting PlAMV.•RT-ddPCR assay was used for detecting PlAMV infection in lily leaf samples from the field.