The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

in Nematology
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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 (R2=0.98; slope = −3.38; E=97.6%). 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.

The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

in Nematology

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References

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Figures

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    A, B: Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 62°C and 63°C showing multiple peaks of two Pratylenchus thornei populations (n=3) and NTC (n=2); C, D: Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 65°C and 68°C showing multiple peaks of two P. thornei populations (n=3) and NTC (n=2).

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    Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 69°C showing high peaks at ±85.5 of two Pratylenchus thornei populations (n=3) and NTC (n=2).

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    Standard curve of the qPCR assay for Pratylenchus thornei threshold cycle number (Ct) plotted against the log of the number of individuals of P. thornei (1, 5, 10, 20, 40) (n=3).

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