Development and validation of four TaqMan real-time PCR diagnostics for the identification and quantification of Pratylenchus crenatus, Pratylenchus neglectus, Pratylenchus penetrans and Pratylenchus thornei

Abstract

Pratylenchus crenatus, P. neglectus, P. penetrans and P. thornei are globally the most commonly occurring species of root-lesion nematodes (RLN). Correct identification and quantification of these nematodes is important for strategic management interventions such as rotation choice and nematicide use. A real-time quantitative PCR can provide a fast and reliable alternative to morphological identification, which requires significant taxonomic experience. A TaqMan hydrolysis probe method based on the 28S rDNA D2-D3 expansion region was developed and validated for the identification and quantification of these four root-lesion nematode species. Standard curves for each target RLN species were generated by plotting known gene copy number, obtained by a ten-fold serial dilution of purified plasmids, with corresponding Ct values. Each standard curve had a strong linear correlation () between Ct value and gene copy number. There was consistent amplification of samples with target species from different geographic locations within the UK, whereas a lack of amplification was noted for selected non-target species: P. coffeae, P. pseudocoffeae, P. vulnus, P. fallax, Globodera rostochiensis, Meloidogyne hapla, Trichodorus primitivus and Bitylenchus hispaniensis. Specificity and sensitivity of the methods were confirmed by three experiments that explored different life stages, increasing the number of target species, and had mixed Pratylenchus samples. Finally, estimates obtained by qPCR methods were compared with counting carried out by microscopy showing a good correlation (). The TaqMan real-time PCR developed in this study provides a specific, fast and accurate quantification of P. crenatus, P. neglectus, P. penetrans and P. thornei.