Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan Probe and SYBR Green real-time PCR assays

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The ectoparasitic stubby root nematode, Paratrichodorus allius, transmits tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. A diagnostic method for direct quantification of P. allius from soil DNA using TaqMan probe and SYBR Green real-time PCR assays was developed to assist the potato industry in management of this important vector. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA of P. allius was demonstrated by in silico analysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculated P. allius individuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil. Moreover, the numbers of P. allius determined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented positive correlation relationships (R2=0.80 and 0.86). Although the quantification using TaqMan probe overestimated the target nematodes compared to using SYBR Green in eight out of ten field soil samples, results of the two methods correlated well (R2=0.92). This is the first report of P. allius quantification from soil DNA extracts using real-time PCR, providing a rapid and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification.


International Journal of Fundamental and Applied Nematological Research



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  • Standard curves of serial five-fold dilutions of soil DNA representing an equivalent of 20 to 2.56 × 10−4 individuals in 0.5 g of autoclaved soil. The circle and diamond markers represent individual values for each dilution and replicate. The black and dashed lines present the linear regression generated by TaqMan probe and SYBR Green real-time PCR, respectively.

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  • Relationship between the numbers of Paratrichodorus allius nematodes quantified by real-time PCR assays according to the generated standard curves and the numbers added to 0.5 g of autoclaved soil (0.5, 1, 2, 5, 10 and 15).

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  • Conventional PCR using universal primers rDNA2/rDNA1.58S for detection of DNA presence in the DNA extracts of potato field soil samples in Table 4. M = 100 bp DNA ladder. N = negative control which DNA was extracted from autoclaved soils. S1-S17 represent the DNA extracted from field samples 1-17. 1, 2 and 3 indicates the biological replicates for each soil DNA. H2O = no-template control using ddH2O instead of DNA in a PCR reaction. P = positive control of pure Paratrichodorus allius DNA.

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  • Relationship between the numbers of Paratrichodorus allius nematodes quantified by real-time PCR assays according to the generated standard curves and the numbers determined by manual nematode extraction and microscope method in 200 g of soil from potato field samples.

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  • Relationship between the numbers of Paratrichodorus allius nematodes quantified by TaqMan probe and SYBR Green real-time PCR assays.

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