Automated high-throughput extraction system of sRNA and high-resolution melting quantitative PCR (HRM-qPCR) analysis for viroid detection

A rare viroid disease, Orange Spotting (OS), has been associated with Coconut cadang-cadang viroid variant (named OS-CCCVd) in oil palm. The low concentration of OS-CCCVd in oil palm leaf tissues makes it tedious to obtain high-quality RNA. Various conventional extraction protocols are available for extracting RNA; however, the bottleneck to the methods is the acquisition of good yields of high-quality RNA suitable for use in viroid detection. Studies looking into the automation of magnetic bead extraction systems for viroid detection in oil palm are limited. In this study, we have compared four extraction methods, namely the MagMAX™ mirVana Total RNA isolation kit (Mag-A), MagMAX™ plant RNA isolation kit (Mag-B), modification of MagMAX™ mirVana Total RNA isolation kit (Mag-Mod) and the convention method (NETME buffer). The KingFisher Flex System uses a 96-well plate format for the three automated approaches. The major modification in the Mag-Mod protocol is the inclusion of lithium chloride solution and NETME buffer to the lysis buffer (which enhances RNA recovery) and the reduction in the volume of reagents used per reaction and run conditions, including time. High-quality small RNA was produced as a result of altering the buffers and reagent volume in the Mag-Mod method, which also increased sample productivity (48 samples per day) and detection sensitivity. These effects indirectly decreased the number of technical replicates in a run. Importantly, the alteration permitted the use of ground plant tissue samples with a small sample amount of 0.05- 0.1 g as opposed to the 2-4 grequired with the conventional method. The development of the modified Mag-Mod method using the 96-deep well plate with a magnetic bead system vastly increased our sample throughput, complementing the detection methods (e.g. RT-PCR and HRM-qPCR). Aside from developing an improved extraction method (Mag-Mod method), a novel HRM-qPCR assay successfully differentiated OS-CCCVd variants and identified the presence of OS-CCCVd in samples, obviating the need for Sanger Sequencing.