A real-time quantitative PCR assay was developed for the accurate detection and quantification of the root-lesion nematode, Pratylenchus thornei. A qPCR primer set, including two primers and a probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimised by using the primers with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 62 to 69°C. Based on the Ct values, we retained the program with an annealing temperature of 69°C. The specificity of the qPCR assay including the probe was confirmed by the lack of amplification of DNA from 47 populations belonging to 15 other Pratylenchus species and nine isolates from P. thornei. The assay was very sensitive as it was able to detect a single individual of P. thornei, even when mixed with up to 80 individuals of P. penetrans. DNA was extracted from exactly 80 P. thornei individuals. A dilution series from this DNA resulted in a standard curve showing a highly significant linearity between the Ct values and the dilution rates (; slope = −3.38; ). The qPCR assay developed in this study proved to be specific and sensitive, thus providing a fast and accurate tool for detection and quantification of this pathogen during research, as well as for diagnostic labs.
Detection and quantification of root-knot nematode (Meloidogyne javanica), lesion nematode (Pratylenchus zeae) and dagger nematode (Xiphinema elongatum) parasites of sugarcane using real-time PCR.
Molecular and Cellular Probes22168-176.
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Identification of Pratylenchus thornei, the cereal and legume root-lesion nematode, based on SCAR-PCR and satellite DNA.
European Journal of Plant Pathology118115-125.
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Comparison of the sequences of the D3 expansion of the 26S ribosomal genes reveals different degrees of heterogeneity in different populations and species of Pratylenchus from the Mediterranean region.
European Journal of Plant Pathology111949-957.
De LucaF.ReyesA.TroccoliA.CastilloP. (2011).
Molecular variability and phylogenetic relationships among different species and populations of Pratylenchus (Nematoda: Pratylenchidae) as inferred from the analysis of the ITS rDNA.
European Journal of Plant Pathology130415-426.
HoltermanM.van der WurffA.van den ElsenS.van MegenH.BongersT.HolovachovO.BakkerJ.HelderJ. (2006).
Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades.
Molecular Biology and Evolution231792-1800.
Quantitative detection of the potato cyst nematode, Globodera pallida and the beet cyst nematode, Heterodera schachtii, using real-time PCR with SYBR Green I dye.
Molecular and Cellular Probes1981-86.
Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences.
Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita.
Journal of Plant Nutrition and Soil Science5472-76.