Location: Emerging Pests and Pathogens ResearchTitle: Isolation of nematode DNA from 100 grams of soil using Fe3O4 super paramagnetic nanoparticles
|GORNY, ADRIENNE - Cornell University|
|HAY, FRANK - Cornell University|
|PETHYBRIDGE, SARAH - Cornell University|
Submitted to: Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2017
Publication Date: 1/8/2018
Citation: Gorny, A., Hay, F., Wang, X., Pethybridge, S. 2018. Isolation of nematode DNA from 100 grams of soil using Fe3O4 super paramagnetic nanoparticles. Nematology. 20:271-283.
Interpretive Summary: Plant-parasitic nematodes cause significant yield reductions to a wide range of crops worldwide. Control of plant-parasitic nematodes often relies on chemical treatments without knowledge of nematode presence or population levels. These chemical treatments may be unnecessary if nematode population is insufficient to cause economic damage. In this study, a high-throughput and economical method for extracting DNA to facilitate detection and quantification of nematodes from a large volume of soil was developed. This method utilizes enzymatic laundry detergent for tissue lysis and nucleic acid capture and separation using special nanoparticles. The developed method produces high quality DNA that allows a detection limit of 1 nematode per 1 gram of soil. This highly sensitive soil testing method may aid in molecular diagnostic detection and quantification of nematode and other soilborne pathogens and be adopted as a DNA-based solution for quantifying disease risk to enable information driven management decisions.
Technical Abstract: Soilborne plant-parasitic nematodes cause losses through yield reductions and damage to a broad range of crops worldwide. Control of plant-parasitic nematodes often relies on application of fumigants or non-fumigant nematicides without knowledge of the presence of the nematodes or population densities due to the time lag in obtaining this information through soil sampling, extraction, and counting. A high throughput, economical method for extracting DNA was developed to facilitate detection and quantification of nematodes from 100 g of soil. The assay was tested using the Northern root-knot nematode, Meloidogyne hapla. The method utilized enzymatic laundry detergent lysis, Fe3O4 super paramagnetic iron oxide nanoparticle (SPION) capture, and polyvinylpolypyrrolidone (PVPP) DNA purification (hereafter referred to as SPION capture method). The SPION capture method was compared to a standard phenol-based extraction and to a commercially available DNA extraction kit (MoBio PowerLysis PowerSoil) using 0.5 g of soil. Resultant DNA was approximately 100-fold less but of similar quality to DNA obtained from the standard phenol procedure and the commercial kit. The binding capacity of the SPION capture method was assessed by evaluating DNA yield and quality through increasing SPION volumes, from 1 mg to 50 mg of nanoparticles. Ten mg of nanoparticles was identified to maximize DNA yield while minimizing co-capture of contaminants when extracting from 100 g of soil. Sensitivity was evaluated by assaying samples inoculated with 1, 10, 100, and 1,000 second-stage juveniles of M. hapla per 100 g of soil. The detection limit of the SPION capture method was approximately 100 nematodes per 100 g soil. The SPION capture method extracted M. hapla DNA from mineral soils but would need further optimization for extraction from high organic matter (i.e. ‘muck’) soils. Benefits of this method compared to alternative techniques include improved consistency in resultant DNA yield, reduced processing time and cost, and the use of non-hazardous materials and generic laboratory equipment. The ability to extract DNA from 100 g volumes of soil at a detection limit of 1 nematode per 1 g soil provides advantages in overcoming sampling deficiencies when quantifying populations that are highly spatially aggregated, to enable site-specific control of plant-parasitic nematodes. This method may aid in molecular diagnostic detection and quantification of plant-parasitic nematodes and other soilborne diseases.