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Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR

Mokrini, Fouad, Waeyenberge, Lieven, Viaene, Nicole, Abbad Andaloussi, Fouad, Moens, Maurice
European journal of plant pathology 2013 v.137 no.2 pp. 403-413
DNA, Pratylenchus penetrans, annealing, crops, extension education, farmers, genes, pest management, quantitative polymerase chain reaction, root lesion nematodes, taxonomy, temperature
Pratylenchus penetrans is one of the most economically damaging plant-parasitic nematodes and is found on a wide variety of crops. Correct identification and quantification of this nematode are necessary for providing advice to farmers, but are not easily obtained with the traditional way of microscopic observation. We developed a qPCR assay to detect and quantify P. penetrans in a short but accurate manner. A qPCR primer set, including two primers and a TaqMan probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimized by using the primers in a qPCR assay with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 60 °C to 64 °C. Based on the Ct-values, we retained the program with an annealing temperature of 63 °C. The assay with the probe was very sensitive as it was able to detect a single individual of P. penetrans, even when mixed with up to 80 individuals of P. thornei. The specificity of the reaction was confirmed by the lack of amplification of DNA from 28 populations of 18 other Pratylenchus species and from plant-parasitic nematodes from nine other genera. DNA from 21 different isolates from P. penetrans was amplified. DNA extraction from 80 individuals and quantification by qPCR was repeated four times; Ct-values showed consistent results (Ct = 24.4 ± 0.4). A dilution series from DNA of P. penetrans resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R² = 0.99; slope = −3.23; E = 104 %). The tests showed a high correlation between the real numbers of nematodes and the numbers detected by the qPCR. The developed qPCR assay provides a sensitive means for the rapid detection and reliable quantification of individuals of this pest. This method does not require expertise in nematode taxonomy and morphology, and can be used as a rapid diagnostic tool in research, as well as in diagnostic labs and extension services advising farmers for pest management.