Early Detection of the Root-Knot Nematode Meloidogyne hapla Through Developing a Robust Quantitative PCR Approach Compliant With the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines
Résumé
Root-knot nematodes (RKNs) are major threats to crops through attacking the roots, which induces an abnormal development of the plant. Meloidogyne hapla Chitwood is of particular concern as it is currently expanding its distribution area and displays a wide host range. Effective plant protection against this RKN requires early detection as even a single individual can cause severe economic loses on susceptible crops. Molecular tools are of particular value for this purpose and among them, qPCR presents all the advantages, i.e. sensitivity, specificity, rapidity of diagnosis at a reduced cost. Although few studies already proposed detecting M. hapla through this technique, they lack experimental details and performance testing, and suffered from low taxonomic resolution and/or required expensive hydrolysis probes. Here we propose a qPCR detection method that uses SybrGreen with developed primers amplifying a fragment of COI mitochondrial region. The method is developed and evaluated following the MIQE guidelines to ensure its quality, i.e. sensitivity, specificity, repeatability, reproducibility, robustness. The results demonstrate that the newly developed method fulfills its goals as it proved specific to M. hapla and allowed for a reproductible detection level as low as 1.25 equivalent of a juvenile individual. All criteria associated with the MIQE guidelines were also met, what makes it of general use for the reliable early detection of M. hapla.