POSTERS: Pathogen detection, quantification and diagnosis
Evaluation of factors associated with real-time PCR quantification of the stubby root nematode Paratrichodorus allius from field soil DNA
Guiping Yan - North Dakota State University, Department of Plant Pathology. Danqiong Huang- North Dakota State University, Department of Plant Pathology
The stubby root nematode Paratrichodorus allius is a prevalent vector that transmits Tobacco rattle virus causing corky ringspot disease of potato in the United States. Factors relating to SYBR Green quantitative real-time PCR (qPCR) quantification of P. allius using soil DNA were assessed. Soil pre-treatments showed that autoclaved field soil significantly reduced the DNA amount and quality, and air or oven dried soil yielded a lower amount of DNA with a similar purity compared with natural field soil. Loamy sand soils from potato fields were used. PCR inhibitors were detected in soil DNA without additional purification targeting pBluescript II SK(+)- plasmid DNA, and Al(NH4)(SO4)2 treatment significantly reduced the inhibitors compared with polyvinylpolypyrrolidone column. PCR inhibitors on qPCR were suppressed by bovine serum albumin used as a PCR enhancer. The largest nematode individuals produced significantly lower Cq values than the smallest individuals. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when a grid sampling strategy was used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA with known nematode numbers, produced similar quantification correlation and amplification efficiency. This study will be useful for the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA to improve detection efficiency and accuracy.