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Abstract
To compare the sensitivity of High-Fidelity (Hi-Fi) and standard PCR in detecting
plant pathogens in symptomatic host plant tissue, four DNA extraction methods were tested in
conjunction with a standard and two Hi-Fi PCR protocols. The DNA extraction methods were: 1)
Extract-N-Amp Plant Kit (Sigma-Aldrich); 2) DNeasy Plant Mini Kit (Qiagen), 3) CTAB buffer,
and 4) lithium chloride Shorty buffer. Symptomatic tissues (i.e. leaf, petiole and root tissue) from
selected diagnostic samples were submitted to each extraction method. DNA samples were then
used for each PCR protocol applying species-specific primers: 1) Standard PCR; 2) Hi-Fi PCR
using LongAmp enzyme; and 3) Hi-Fi PCR Taq+Accuzyme. DNA quantification using
spectrophotometry indicated Extract-N-Amp and Shorty methods yielded the highest DNA
amounts with lower purity. Both Hi-Fi PCR protocols were more sensitive than standard PCR. The
Accuzyme protocol detected targeted plant pathogens in all samples using the DNeasy and Extractn-
Amp methods, whereas the standard protocol detected the pathogen only in leaf samples by
using the DNeasy kit. This study demonstrates that Hi-Fi PCR provides a highly sensitive tool for
molecular diagnostics in planta, and that the DNA extraction method influences PCR sensitivity.