1. Academic Validation
  2. Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification

Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification

  • Plant Dis. 2021 Oct;105(10):2867-2872. doi: 10.1094/PDIS-12-20-2730-RE.
Yi-Wen Tseng 1 Chien-Fu Wu 1 Chia-Hwa Lee 1 2 Chung-Jan Chang 1 3 Yuh-Kun Chen 1 Fuh-Jyh Jan 1 2 4
Affiliations

Affiliations

  • 1 Department of Plant Pathology, National Chung Hsing University, Taichung 40227, Taiwan.
  • 2 PhD Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung 40227, Taiwan.
  • 3 Department of Plant Pathology, University of Georgia, Griffin, GA 30223, U.S.A.
  • 4 Advanced Plant Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan.
Abstract

A number of viruses and viroids infect solanaceous Plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous Plants using one-step Reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and Plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from 1 fg to 10 ng, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR, but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability to screen a large number of solanaceous Plants and seeds intended for import and export.

Keywords

RT-LAMP; Solanaceae; pospiviroid; quarantine; universal primer; vegetables; viruses and viroids.

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