Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Abstract

Effective pathogen detection is essential for plant disease control. However, plant sample preparation for downstream assays, such as quantitative polymerase chain reaction (qPCR), is challenging to perform outside of a laboratory. This paper reports two sample preparation methods featuring chemical and mechanical lysis and nucleic acid extraction using a micro-homogenizer, followed by serial dilution or nucleic acid purification with a paper disk before assay. Five minutes of lysis and extraction resulted in DNA and RNA yields of up to 76.5% and 63.3%, respectively, compared to mortar and pestle controls. Crude lysates were unsuitable for direct use in qPCR assays; however, serial dilution or quick wash using chromatography paper rendered samples ready for such assays. Additionally, the nucleic acids stored on paper disks under various storage conditions remained stable for one month. These methods can facilitate the in-field preparation of citrus samples and allow for both onsite and mail-in diagnostics for growers.

Keywords

• sample preparation
• nucleic acid extraction
• micro-homogenizer
• paper disk
• citrus diseases