Assessment\nof Real-Time PCR Techniques for the Detection\nof Airborne Fungal Pathogens of Wheat: Role of DNA Extraction on Spore\nQuantification

Our research focused\non developing a highly sensitive\nwhole-spore\nreal-time immuno-PCR (RT-iPCR) assay for the detection of three wheat\nfungal pathogens: Pyrenophora tritici-repentis (<i>Ptr</i>), Fusarium graminearum (<i>Fg</i>), and Puccinia striiformis forma specialis (f. sp.) <i>tritici</i> (<i>Pst</i>). RT-iPCR measurements were compared to more well-established quantitative\nPCR (qPCR) assays to compare their performance. While specificity\nremained a challenge for RT-iPCR, the direct spore measurements negate\nthe need for DNA extraction, making RT-iPCR a potentially valuable\ntechnique that warrants further research. An alternative approach\nwas developed to determine DNA extraction efficiency and quantification\nof spore numbers by qPCR, which is currently a methodological gap\nin most qPCR spore measurements. DNA extraction efficiency determined\nfor <i>Fg, Pst</i>, and <i>Ptr</i> spores were\n5.0 ± 0.1, 7.0 ± 0.4, and 290 ± 36%, respectively,\ndemonstrating important implications for the accuracy of these techniques\nwhen DNA recovery is not considered.