Application of Rapid and Reliable Detection of Cymbidium Mosaic Virus by Reverse Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Immunoassay

Abstract

Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39°C) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.

Development and Application of a Duplex RT-RPA Assay for the Simultaneous Detection of Cymbidium mosaic virus and Odontoglossum ringspot virus

“…Sun developed a TaqMan probe-based triplex RT-qPCR protocol for detecting CymMV, ORSV, and CymRSV simultaneously [ 14 ]. Several of these assays typically involve costly equipment, extended durations, and rigorous operating specifications [ 41 ].…”

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses

“…Its isothermal nature renders it suitable for resource-limited settings, and the relatively short reaction time positions it as a valuable tool for rapid DNA amplification. By incorporating specially modified probe primers into the amplification system and collaborating with LFT, RPA-LFT has emerged as a primary method for diagnosing plant viruses with varied genome types, encompassing +ssRNA, negative sense (−) ssRNA, ambisense RNA (±RNA), dsRNA, ssDNA, dsDNA, and even viroids with naked circle RNA [1,7,[18][19][20][21][22][23][24][25][26][27][28][29][30] (Table 1). Rice stripe mosaic virus -ssRNA 30 min 10 7 dilution [38] Tomato brown rugose fruit virus +ssRNA 20 min 10 1 copies /reaction [39] Pepper mild mottle virus +ssRNA <1 h - [40] Tomato mosaic virus [40] Tomato mottle mosaic virus [40] *: the data are for reference only since they are obtained directly from the literature, where different references (total DNA/RNA, viral DNA/RNA, dilution fold, etc.)…”

“…Recently, several other +ssRNA genome viruses have been targeted for detection by RPA-LFT, including cymbidium mosaic virus (CymMV) in the genus Potexvirus of the family Alphaflexiviridae, barley yellow dwarf virus (BYDV) in the genus Polerovirus of the family Solemoviridae, cowpea mild mottle virus (CPMMV) in the genus Carlavirus of the family Betaflexiviridae, actinidia chlorotic ringspot-associated virus (AcCRaV) in the genus Emaravirus of the family Fimoviridae, and bean pod mottle virus (BPMV) in the genus Comovirus of the family Secoviridae [21][22][23]26,28]. The developed RPA-LFT assay for these viruses exhibited 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR), providing a simple, rapid, sensitive, and reliable method for viral diagnosis in the field.…”

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for Detection of Plant Viruses

“…Its isothermal nature renders it suitable for resourcelimited settings, and the relatively short reaction time positions it as a valuable tool for rapid DNA amplification. By incorporating specially modified probe primers into the amplification system and collaborating with LFT, RPA-LFT has emerged as a primary method for diagnosing plant viruses with varied genome types, encompassing +ssRNA, negative sense (-) ssRNA, ambisense RNA (±RNA), dsRNA, ssDNA, dsDNA, and even viroids with naked circle RNA [1,6,[18][19][20][21][22][23][24][25][26][27][28][29][30] (Table 1). Little cherry virus 2 (LChV2), belonging to the genus Ampelovirus in the family Closteroviridae with a +ssRNA genome, causes little cherry disease (LCD) in sweet cherries (Prunus avium) globally.…”