Application Of High Fidelity Pcr In The Plant Disease Diagnostic Lab

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.