NEMATOOLS diagnostic assays: development and validation of real-time PCR for detection and identification of Meloidogyne chitwoodi and M. fallax

Summary Root-knot nematodes Meloidogyne chitwoodi and M. fallax , major soil-borne pathogens of potato, are listed as quarantine pests in the European Union. This classification requires the use of rapid and reliable tools for monitoring such organisms and thus preventing their potential introduction and spread. In this study, we developed and validated two specific and sensitive real-time polymerase chain reaction (PCR) assays in order to identify these two species from isolated individuals and to detect them in soil extracts. The two primer pairs designed, McN and MfN, targeting species-specific regions of the intergenic spacer (IGS) rDNA locus, enabled the amplification of DNA of all the target populations tested and no cross-reaction was observed when tested against a large number of non-target phytoparasitic nematodes. The identification assay was highly sensitive, being effective at a threshold of one female and one second-stage juvenile (J2) and was not impacted by the presence of other nematodes in the samples. The detection assay was able to reliably detect five J2 of M. chitwoodi or M. fallax in a soil extract and is usable in duplex format, thus allowing the simultaneous detection of both species. Our detection assay was also validated with the incorporation of a heterologous internal amplification control (IAC), thus improving our assay for routine diagnostics. The real-time PCR assays also demonstrated high levels of repeatability and reproducibility and was tested with success on routine samples. These assays have also been the subject of interlaboratory testing, confirming their robustness. These new PCR assays provide rapid and accurate identification and detection tools for use in diagnostic laboratories.