Validation of NA extraction internal control procedures for diagnosis

Plant health labs routinely implement a high diversity of nucleic acid extraction methods, driven by factors such as the organism, test purpose, and matrix, which all have their specific characteristics, e.g. inhibitor presence. However, this diversity in factors and the lack of standardized procedures in plant health results in varied methodologies, including in-house and published approaches. Furthermore regulated disease diagnostics has evolved considerably in recent years to rely essentially on molecular diagnostics. In this context, the lack of a nucleic acid quality control procedure poses a risk to diagnostic reliability. EPPO recognised the need to evaluate the NA extraction process as an important step in routine diagnostics and this resulted in a project (accronym IVENAD), engaging over 30 labs, seeks to inventory and validate quality control procedures for nucleic acid extraction in plant pest diagnosis with a specific focus on internal control procedures, designed to target housekeeping genes within the matrix, allowing for a comprehensive assessment of the extraction process's effectiveness. The aim is to establish guidelines for control procedures of the different extraction methods. A desktop study led to the inventory of a 110 articles and 170 protocols provided by 19 labs. Despite the absence of a uniform methodology, further prioritization and categorization of all the extraction methods and control procedures that the labs are implementing, led to a selection of RNA and DNA extraction methods on 4 different matrices (3 plant and 1 insect) to undergo evaluation against a variety of 21 internal controls, specifically targeting a variety of matrix housekeeping genes. This selection underwent a series of extraction tests in view of a further selection of matrix-target and control combinations that will later be included in a test performance study (TPS) organised among the project partners. In addition, the project wants to deliver a set of (partially) validated nucleic acid extraction procedures, both for RNA and DNA applications (e.g. virus and bacteria detection) in (complex) matrices. The results in turn can generate some useful guidelines and standardize procedures benefitting National Plant Protection Organizations and diagnostic labs. The implementation of reliable routine nucleic acid extraction procedures as part of the diagnostic procedures enhances rapid and reliable pathogen/pest detection, enabling timely intervention and preventing potential spread.