Efficient quantification of Globodera pallida and G. rostochiensis (Tylenchida: Heteroderidae) in large amounts of soil using probe-based real-time PCR

Abstract

Real-time quantitative polymerase chain reaction (qPCR) is used to estimate the population densities of the potato cyst nematodes Globodera pallida Stone and Globodera rostochiensis (Wollenweber) Skarbilovich (Tylenchida: Heteroderidae). Since it is difficult to extract nematode DNA from large amounts of soil (≥ 100 g, enough for quantification of cyst nematodes), cyst isolation is required before DNA extraction. However, when isolating cysts from the soil, various impurities are simultaneously isolated, and separating the cysts from these impurities is laborious. Although previous studies have reported methods for extracting DNA from mixtures of cysts and impurities, it is unclear whether such DNA can be used to estimate nematode densities using qPCR. To examine the effects of impurities on the accuracy of qPCR quantification, we extracted DNA from nematode eggs (G. pallida and G. rostochiensis) mixed with impurities and performed qPCR. The results suggested that the differences in the fields affected the quantification accuracy. Therefore, field-specific standard curves should be set, which are impractical for routine diagnosis. To propose a more practical method, we determined a fixed standard curve for each species and estimated the population densities in field soil samples by qPCR using the standard curves. The estimated population densities significantly correlated with those determined using conventional microscopic inspections. This study revealed that the population densities of G. pallida and G. rostochiensis can be estimated from large amounts of soil, probably only approximately, but efficiently, by qPCR using DNA extracted from mixtures of cysts and impurities.