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R. CAMPOS-HERRERA ET AL.
they perform well with material extracted from soil. When DNA was isolated
directly from soil, the PowerMax Soil TM DNA Isolation Kit (MoBio) was more
reliable than a standard laboratory method or the Ultra Clean Soil TM DNA Kit
(MacMillan et al., 2006). For nematodes extracted from soil, Madami, Subbotin, and
Moens, (2005) concluded that the use of proteinase K followed by PCR Buffer
yielded more DNA than if it was followed by Worm Lysis Buffer. Donn et al.
(2008) found that use of sodium hydroxide extraction, ChargesSwitch PCR CleanUp
Kit (Invitrogen), QIAquick PCR Purification System (Invitrogen) and the Wizard
PCR Prep DNA Purification System (Promega) all failed to provide either enough
DNA or DNA of high enough quality for PCR whereas phenol chloroform
extraction or Purelink PCR Purification Columns (Invitrogen) provided high
concentrations of DNA acceptable for PCR studies. Campos-Herrera et al. (2009)
obtained good quality DNA from nematodes extracted with sucrose-centrifugation
using the Unltra Clean Soil TM DNA Kit (Mo Bio).
PCR can be inhibited by chemical contaminants in soil (e.g., humic acid,
phenolic compounds) or by use of excessive template. Inhibition can be complete or
partial, and so it is important to calibrate qPCR with results obtained from known
quantities of the target organism (e.g., nematodes, nematodes infected by NF, etc.)
added to populations of nematodes extracted from soil rather than relying on PCR of
DNA from pure cultures. Bovine serum albumin has been used to reduce the effects
of chemical inhibitors of PCR (MacMillan et al., 2006; Torr et al., 2007; Campos-
Herrera et al., 2009). To avoid excess template, dilution of DNA from 4 to 100-fold
from samples of unknowns is usually necessary (Madami et al., 2005; Jones, Todd,
& Herman, 2006; Kang et al., 2009). Because each soil sample yields a different
quantity of DNA that might affect the reaction, the use of a standard DNA quantity
is preferable to a standard dilution.
Most authors report very high detection efficiency from the use of qPCR, which
is frequently at the level of a single nematode. Several multiplex systems in which
single reactions measure more than one nematode species or combinations of
nematodes and fungi have been developed that can substantially reduce time and
cost (Madami, Ward, & de Boer, 2008; Jones et al., 2006; Berry, Fargette, Spaull,
Morand, & Cadet, 2008; Zijlstra & van Hoof, 2006). Unfortunately, the increased
likelihood of competition between target DNA or development of interacting
primers (primer-dimers) that increase florescence for a false signal can impede the
development and performance of multiplex systems. Zijlstra and van Hoof (2006)
reported a density-dependent reduction in the efficiency of multiplex qPCR and
suggested that individual reactions would be required for precise estimation. Berry
et al. (2008) observed similarity of melting temperatures and competition between
the amplification of Meloidogyne javanica and Pratylenchus zeae targets, which
made it impossible to distinguish the two species in a multiplex reaction.
At present, studies using molecular techniques to assess EPN population
distribution and dynamics remain rare but the development and optimization of
these techniques could contribute much to our understanding of these nematodes and
the kinds of strategies that might facilitate their enhanced usefulness for biological
control in agroecosystems.