Isolation of nematode DNA from 100 g of soil using Fe3O4 super paramagnetic nanoparticles

in Nematology
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An economical method for extracting nematode DNA from 100 g of soil was developed to facilitate nematode detection and quantification, and tested using the Northern root-knot nematode, Meloidogyne hapla. The method utilised enzymatic laundry detergent lysis, Fe3O4 super paramagnetic iron oxide nanoparticle (SPION) capture, and polyvinylpolypyrrolidone (PVPP) purification. Resultant DNA from this SPION capture method was approximately 100-fold less but of similar quality to DNA obtained from a standard phenol procedure and a commercial DNA extraction kit. An addition of 10 mg of nanoparticles to the extraction lysate was identified to maximise DNA yield while minimising co-capture of contaminants. The detection limit of the SPION capture method was approximately 100 nematodes (100 g soil)−1. The SPION capture method extracted nematode DNA from mineral soils but requires further optimisation for extraction from high organic matter (i.e., ‘muck’) soils. The benefits of this method compared to alternative techniques are discussed.

Isolation of nematode DNA from 100 g of soil using Fe3O4 super paramagnetic nanoparticles

in Nematology

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References

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Figures

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    A: Genomic DNA isolated from Meloidogyne hapla second-stage juveniles using each extraction method was visualised on an agarose gel stained with GelRed. M: 15 ng phage lambda DNA, molecular weight 48 kb; Lanes 1 and 2: SPION capture method; Lanes 3 and 4: conventional phenol method; Lanes 5 and 6: commercial kit extraction; B: Resultant DNA from each method was PCR amplified using M. hapla species-specific primers (Hay et al., 2016) targeting a segment of the internal transcribed spacer (ITS) region. Amplification products are approximately 150-bp in size (arrowhead). M: 100-bp DNA ladder; Lanes 1 and 2: SPION capture method; Lane 3: SPION capture method negative control; Lanes 4 and 5: conventional phenol method; Lane 6: phenol method negative control; Lanes 7 and 8: commercial kit extraction; Lane 9: commercial kit extraction negative control; Lane 10: positive PCR control amplified from M. hapla.

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    A SPION-based DNA extraction method was performed on 0.5 g of soil and compared to a standard phenol method and a commercial kit. Each soil sample was inoculated with ten Meloidogyne hapla second-stage juveniles from an inoculum solution and air dried. Ten replicates of each method were performed and the experiment was repeated. Box-plots were generated from the data to evaluate variability in the (A) DNA yield (pg μl−1), (B) A260/280 ratio and (C) A260/230 ratio. Within each plot, the data median is represented by the bold line. Upper and lower quartiles are represented by the edges of the box. Box whiskers denote maximum and minimum values, and dots mark outliers.

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    A: Addition of increasing amounts of super paramagnetic iron oxide nanoparticle (SPION) from 1-50 mg had no significant effect on DNA yield from Meloidogyne hapla second-stage juveniles. Bars in the figure represent the standard error of the mean. B: Resultant DNA from each SPION volume trial was PCR amplified using M. hapla species-specific primers (Hay et al., 2016). Amplification products are approximately 150-bp in size (arrowhead). Lanes 1 and 2: 1 mg SPION; Lanes 3 and 4: 5 mg SPION; Lanes 5 and 6: 10 mg SPION; Lanes 7 and 8: 20 mg SPION; Lanes 9 and 10: 50 mg SPION; Lane 11: extraction negative control; Lane 12: positive PCR control amplified from M. hapla; M: 100-bp DNA ladder. Consistent amplification is noted at the addition of 10 mg of nanoparticles.

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    A: Increasing the number of inoculated nematodes resulted in an increasing DNA yield isolated using 100 g-SPION capture method from soil samples inoculated with 1, 10, 100 or 1000 Meloidogyne hapla second-stage juveniles. However, only the 1000 nematodes per 100 g soil was significantly greater than the population densities. Bars in the figure represent standard error of the mean; B: Resultant genomic DNA (15 μl) was separated on a 1% agarose gel stained with GelRed. Lanes 1 and 2: 1 nematode (100 g soil)−1; Lanes 3 and 4: 10 nematodes (100 g soil)−1; Lanes 5 and 6: 100 nematodes (100 g soil)−1; Lanes 7 and 8: 1000 nematodes (100 g soil)−1; M: 15 ng phage lambda DNA.

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    Resultant DNA from sensitivity testing of the 100 g-SPION capture method was PCR amplified using Meloidogyne hapla species-specific primers (Hay et al., 2016). Products are approximately 150-bp in size (arrow head). Lanes 1 and 2: 1 nematode (100 g soil)−1; Lanes 3 and 4: 10 nematodes (100 g soil)−1; Lanes 5 and 6: 100 nematodes (100 g soil)−1; Lanes 7 and 8: 1000 nematodes (100 g soil)−1; Lane 9: extraction negative control; Lane 10: positive PCR control amplified from M. hapla; M: 100-bp DNA ladder.

  • View in gallery

    Resultant DNA from the 100 g-SPION capture method performed with two mineral soil samples was PCR amplified using Meloidogyne hapla species-specific primers (Hay et al., 2016). Products are approximately 150-bp in size (arrow head). Lanes 1 and 2: two representative samples from Mineral Soil 1; Lane 3: negative extraction control from Mineral Soil 1; Lanes 4 and 5: two representative samples from Mineral Soil 2; Lane 6: negative extraction control from Mineral Soil 2; Lane 7: positive PCR control amplified from M. hapla; M: 100-bp DNA ladder.

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