An Improved Procedure for Extracting Nucleic Acids from Citrus Tissues for Diagnosis of Citrus Viroids.

Abstract

When detecting viroids in citrus tissues, contaminating polysaccharides and phenolic compounds often make it difficult to consistently extract nucleic acids. To extract the nucleic acids consistently, the method was improved as follows: the contaminants were removed by differential 2-butoxyethanol precipitation instead of a combination of 2-methoxyethanol extraction and cetyltrimethylammonium bromide precipitation. In contrast to the conventional method, the modified one consistently extracted some citrus viroids, i.e., citrus exocortis viroid (CEVd), group I citrus viroid (CVd-I) and hop stunt viroid-citrus isolate, which is a variant of group II citrus viroid (CVd-II), after they were sufficiently subjected to sequential polyacrylamide gel electrophoresis (sPAGE) and dot blot hybridization using digoxigenin (DIG)-labeled cRNA probes. A viroid-like RNA presumed to be a group III citrus viroid (CVd-III) was detected in nucleic acids extracted from Japanese citrus samples by sPAGE. The cDNA fragments of the viroid-like RNA were cloned and sequenced. The nucleotide sequences of the cDNAs were completely identical to those reported previously for CVd-IIIa and CVd-IIIb abroad. CVd-III was detected from several other Japanese citrus samples using the DIG-labeled cRNA probe prepared from the cloned cDNA. Many of these samples were also co-infected with other citrus viroids.