Quantitative real-time PCR analysis of 'Candidatus Phytoplasma mali' without external standard curves
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Apple proliferation caused by 'Candidatus Phytoplasma mali' is an economically important disease of apple (Malus × domestica). The availability of a simple quantitative approach to assess pathogen load in infected host plants would certainly contribute to a better understanding of pathogenesis and epidemiology. This study proposes a quantification approach not requiring the analysis of external standard curves. It is based on the simultaneous detection of the 16S rRNA gene of the pathogen and the 1-aminocyclopropane-1-carboxylate oxidase gene of the host plant in a single-tube reaction using TaqMan chemistry. The quantity of the phytoplasma relative to its host plant is determined as the difference between C values of the two target genes (ΔC ). To assess the agreement of the relative quantification approach with a standard curve-based method, a dataset of 450 DNA samples from infected apple trees was reanalysed. Comparison of the ΔC -based relative quantities with the corresponding absolute values revealed high degrees of agreement between the relative and absolute quantification methods. The ΔC procedure can thus be considered adequate for quantification of the phytoplasma in infected host plant tissue. In addition, this approach ensures improved methodological standardisation and increased analysis throughput. © 2012 The Author(s).
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