Quantitative detection of Pratylenchus penetrans from soil by using soil compaction and real-time PCR

概要

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A combination method consisting of soil compaction and real-time PCR was developed to quantify Pratylenchus penetrans in soil. When five andosols and three gray lowland soils naturally infested with P. penetrans were compacted and used to quantify P. penetrans, the threshold cycle (Ct) values were always lower compared with those in non-compacted soils. Furthermore, Ct values were not obtained in some cases and standard errors were sometimes large. To avoid these problems, a DNA extraction method was newly developed and used to quantify P. penetrans in the two naturally infested soils. We obtained Ct values two to four times lower in the modified method than those in the original method. When known numbers of second-stage juvenile (J2) P. penetrans were added to 20 g samples of non-infested andosols and gray lowland soils, which were then compacted, and DNA was extracted by the modified method, there were highly significant correlations (r⊃2; = 0.993 and r⊃2; = 0.965, respectively) between the Ct values and the number of J2s added. The detection limits for P. penetrans in 20 g of soil were 10 and 25 individuals for gray lowland soil and andosol, respectively. To improve the detection limit, the DNA extraction method should be further modified.
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