Simple template-based reverse transcription-recombinase polymerase amplification assay for routine diagnosis of citrus tristeza virus.

This study reports a simple template-based reverse transcription-polymerase amplification assay (ST-RT-RPA) for detection of citrus tristeza virus (CTV) from crude plant extract lysed in NaOH:EDTA (1:1) without the need of tedious RNA isolation. The developed assay showed versatility in its usage as amplification can be performed at wide temperature range (14°C to 42°C) and incubation time (4 to 32 min), although the best conditions were 38°C for 30 min. The developed ST-RT-RPA assay could detect the CTV up to 10-8 dilution of crude plant extract of NaOH:EDTA and up to 0.01 fg µl-1 of RNA of CTV-infected plant tissues and 0.001 ag µl-1 of plasmid DNA containing viral insert, thus exhibiting sufficient sensitivity. ST-RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus yellow vein clearing virus, and Candidatus Liberibacter asiaticus) and was more sensitive in detection of CTV infection in field samples as compared to standard reverse transcription-polymerase chain reaction (RT-PCR) with later showing false negative in 7.92% of samples tested after 1 week of sampling. The developed ST-RT-RPA assay used minimally processed crude plant extract as template, tolerant to sample degradation in transit and storage, while it can be easily performed at wide temperatures and could be adopted in resource-poor setup.