1. Introduction

The European Union Reference Laboratory for Insects and Mites has to verify the quality of reagents used for the diagnosis of plant pests. In plant health sector, quality determination of reagents or reagent lots is still in an immature stage in particular in the area of molecular diagnosis.

The aim of this task is to provide EU-NRLs a list of approved reagents for molecular identification of insects. Due to the broad range of reagents and DNA extraction procedures used in molecular biology and the several influencing factors on their efficiency, the listing of single reagents and DNA extraction procedures only seems not to be useful at the current stage. The evaluation of reagents and DNA extraction is always linked to particular testing procedures. Therefore, only the full verification reports, including general recommendations, will be made available to the EU-NRLs.

Please find below the following reports

Verification of DNA extraction procedures for insects

Verification of Whole Genome Amplification (WGA) for reference material provision

Verification of PCR master mixes for selected TaqMan real-time PCR tests

2. Verification of DNA extraction procedures for insects

The European Reference Laboratory for Insects and Mites has to verify the quality of reagents used for the diagnosis of plant pests. In plant health sector, quality determination of reagents or reagent lots is still in an immature stage in particular in the area of molecular diagnosis. One task of the working programme 2019/2020 was to evaluate different DNA extraction procedures, frequently used by EU-NRLs and/or recommended in EPPO and IPPC diagnostic standards or scientific literature. The aim of this task is to provide EU-NRLs a list of approved procedures and kits for DNA extraction of insects.

3. Verification of Whole Genome Amplification (WGA) for reference material provision

The European Reference Laboratory for Insects and Mites has to provide reference material to the National Reference Laboratories (NRLs), as part of its official tasks. High quality reference material is necessary for numerous regulated taxa and for usage in various activities, such as proficiency test organisation, hands-on trainings, validation studies or providing it to NLRs. Said reference material exists in two forms, namely morphological specimens and DNA collections
Biological material for DNA extraction is rare and precious. Extraction of enough DNA can be difficult because oftentimes only single insect legs are available, or non-destructive DNA extraction must be performed, to preserve specimens. Whole Genome Amplification is a technology for non‐selective amplification of the whole genome sequence. It makes it possible to obtain a large amount of genetic information from a small amount of DNA and it provides a valuable tool for preserving limited samples in molecular biology. In this study we tested the whole genome amplification (WGA) applicability for selected insect species.

4. Verification of PCR master mixes for selected TaqMan real-time PCR tests

The European Reference Laboratory for Insects and Mites has to select, adapt or develop reliable identification tests for the phytosanitary insect and mite species that are relevant for the European Union (included in the Commission Delegated Regulation (EU) 2019/1702 and in the EURL for Insects and Mites working programmes). One sub-activity of the working program is to perform tests for the verification regarding the quality of reagents used for the diagnosis of plant pests. Reagents such as DNA extraction kits and DNA polymerases/master mixes have the potential to significantly affect the performance of molecular tests.
The primary aim of this study is to conduct a comprehensive verification of TaqMan reagents specifically utilized for the molecular identification of insects and mites. Therefore, this study also aims to ensure the availability of high-quality reagents suitable for the molecular identification of selected organisms. This involves evaluating the performance and suitability of these reagents for identification of selected species, focusing on EU-regulated pests.