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Reverse transcription loop-mediated isothermal amplification (RT-LAMP) designed for fast and sensitive on-site detection of Pepper chat fruit viroid (PCFVd)
- Tangkanchanapas, Parichate, Höfte, Monica, De Jonghe, Kris
- Journal of virological methods 2018 v.259 pp. 81-91
- Pepper chat fruit viroid, RNA, cost effectiveness, detection limit, fruit quality, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, reverse transcription loop-mediated isothermal amplification, tomatoes, trade, viroids, viruses
- Pepper chat fruit viroid (PCFVd) is one of the most important tomato and pepper diseases causing serious losses, affecting productivity, fruit quality and even international seed trade. Reverse transcription Loop-mediated isothermal amplification (RT-LAMP) is a fast and reliable RNA amplification assay, out competing conventional reverse transcription polymerase chain reaction (RT-PCR) in robustness, analytical sensitivity and specificity, and cost-effectiveness. In this work, a PCFVd specific RT-LAMP detection assay, based on a set of six primers was developed. Under the optimized conditions, PCFVd could be detected within 15 min, with a sensitivity of detecting PCFVd that was almost the same as a probe-based qRT-PCR and 10–100 times higher than the available RT-PCR methods. No cross-amplification with other viroids and tomato viruses was observed. The validated assay was also adapted for convenient on-site detection. Besides replacing the full RNA extraction with a simple lysis procedure, several visualization options, of which the use of SYTO9 was the most convenient, were presented to accommodate any in-field application of the method.