Hydrolysis probe-based PCR for detection of Pratylenchus crenatus, P. neglectus and P. penetrans

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Molecular detection of pest and pathogens relies on rapid and dependable methods for their identification as well as an assessment of their abundance. This study describes the development and evaluation of a diagnostic method for detection of Pratylenchus crenatus, P. penetrans and P. neglectus, based on a hydrolysis probe qPCR assay. Primer/probe sets were designed targeting the ITS-1 rDNA. In order to assess the specificity, primer/probe sets were tested with samples of non-target Pratylenchus species and Radopholus similis. Experiments using dilutions of purified plasmid standards tested the sensitivity of the hydrolysis assay against detection of DNA extracted from individual nematodes. Target DNA was detected in soil samples collected from potato fields and this indicated that P. crenatus, P. neglectus and P. penetrans are widely distributed in Scotland, frequently co-existing in mixed populations, with P. crenatus more prevalent than either P. neglectus or P. penetrans.

Hydrolysis probe-based PCR for detection of Pratylenchus crenatus, P. neglectus and P. penetrans

in Nematology

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Figures

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    Standard linear curves of cycle threshold (Ct) values plotted against log-transformed plasmid DNA standards of known concentrations of A: Pratylenchus crenatus; B: P. penetrans; C: P. neglectus. The standard curves were run in duplicate for each Pratylenchus species and the standard error bars represent the differences between different runs.

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    Estimated Pratylenchus spp. populations from 50 Scottish potato fields using microscopy and qPCR (average, standard deviation, minimum and maximum values). A: Nematode abundances of each sample calculated through both methods (P<0.01); B: Number of each Pratylenchus species estimated by qPCR.

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