A positive-control PCR assay that is based on chloroplastndhB sequences and applicable to nucleic acid extractions from diverse plant taxa

Abstract

Diagnostic capacity is a key component of phytosanitary systems designed to exclude or control unwanted pests and diseases. Because of their sensitivity, speed, and application to previously intractable problems, molecular methods are increasingly being used for diagnosis of pests or pathogens with either DNA or RNA genomes. This article describes methods for extracting nucleic acids suitable for PCR-or reverse transcription-polymerase chain reaction (RT-PCR)-based sequence detection from plant species in diverse botanical taxa. A modified hexadecyltrimethylammonium bromide (CTAB) extraction procedure produced total nucleic acid (T-NA) extracts suitable for PCR or RT-PCR from 62 of the 68 species or cultivars tested. For the remaining 8 taxonomically diverse plant species, commercially available DNA extraction kits yielded nucleic acids that were suitable for PCR. Goodquality RNA extractions were obtained from leaf tissue in most cases, by use of either a modified phenol-guanidine isothiocyanate method or proprietary reagents. When examining the suitability of the extraction methods for tissues other than young leaves such as roots, bark, flowers, and rhizomes/bulbs/tubers, a wider range of modifications to the methods was required to obtain suitable T-NA or RNA extractions. The value of PCR and RT-PCR assays based on chloroplastndhB gene sequences as endogenous positive controls was demonstrated by using both RNA and T-NA extracted from a wide range of botanical taxa, including both dicotyledonous and monocotyledonous plants.