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

Our research focused on developing a highly sensitive whole-spore real-time immuno-PCR (RT-iPCR) assay for the detection of three wheat fungal pathogens: Pyrenophora tritici-repentis (Ptr), Fusarium graminearum (Fg), and Puccinia striiformis forma specialis (f. sp.) tritici (Pst). RT-iPCR measurements were compared to more well-established quantitative PCR (qPCR) assays to compare their performance. While specificity remained a challenge for RT-iPCR, the direct spore measurements negate the need for DNA extraction, making RT-iPCR a potentially valuable technique that warrants further research. An alternative approach was developed to determine DNA extraction efficiency and quantification of spore numbers by qPCR, which is currently a methodological gap in most qPCR spore measurements. DNA extraction efficiency determined for Fg, Pst, and Ptr spores were 5.0 ± 0.1, 7.0 ± 0.4, and 290 ± 36%, respectively, demonstrating important implications for the accuracy of these techniques when DNA recovery is not considered.