Optimization of total nucleic acid extraction method for detecting Coconut cadang-cadang viroid variants in oil palm

Abstract

Variants of Coconut cadang-cadang viroid (CCCVd) with over 90% sequence similarity to CCCVd in coconut have recently been associated with orange spotting disease in oil palm, especially in low concentrations in infected hosts. Thus, there is a need to extract high-quality nucleic acid for molecular detection of the viroid. Total nucleic acid (TNA) was extracted from oil palm leaf samples with orange spotting symptoms collected from different states in Malaysia using a modified and optimized version of the conventional natrium chloride EDTA Tris-HCL mercaptoethanol extraction method. The modifications involved additional ethanol and lithium chloride precipitation stages, thereby eliminating the need for further purification through non-denaturing polyacrylamide gel electrophoresis (PAGE). The modified procedure yielded a mean volume of 60 μg RNA per 10 g of fresh tissue which is approximately four times the volume produced by the conventional extraction method. Furthermore, the modified method resulted in higher purity than the conventional method according to the ratios of absorbance at wavelengths of 260/280 nm and 260/230 nm, which were 1.838 and 1.883 with the modified method and 1.085 and 0.765 with the conventional method. The modified method enabled extraction of high-quality RNA from all samples investigated. The extracted RNA was suitable for cDNA synthesis and PCR amplification and showed consistent detection of CCCVd-like RNA at approximately 250 amplicons. Using the conventional method, detection of CCCVd-like RNA was inconsistent and only feasible after the PAGE purification step.