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R. CAMPOS-HERRERA ET AL.
presentation of the methods and results obtained using qPCR. Some of methods
reported to date are reviewed and compared.
Most species-specific primers (and probes, if required) have been developed to
amplify sections of the ITS region. This region of ribosomal DNA is usually well
conserved at the species level but provides greater variability between species than
more highly conserved regions such as D2–D3. Extensive interspecific variation is
necessary to provide enough species-specific primer targets to identify those that
will amplify segments of an optimum size for reliable qPCR reactions (i.e., between
80 and 200 base pairs). Compared to other gene regions, the large ITS database in
GenBank facilitates validating the species-specificity of ITS primers among
organisms characterized to date. In addition to ITS, other regions used to develop
primers/probes for use in qPCR include 18S from rDNA (Holeva et al., 2006;
MacMillan, Blok, Young, Crawford, & Wilson, 2006), MspI satDNA monomeric
unit (François et al., 2007), Hsp70 sequence (Leal et al., 2007) and the intragenic
spacer (IGS) region of the 5S rRNA gene (Kang, Moon, Lee, Shin, & Lee, 2009).
Figure 7. General protocol to develop real time qPCR primers and probes (from
Campos-Herrera, Johnson, El-Borai, Graham, Duncan, 2009).