Rapid-cycle PCR detection of Pyrenophora graminea from barley seed

Sixty RAPD primers were used to screen for a diagnostic marker that could b e used to identify Pyrenophora graminea, a fungal seedborne pathogen that c auses leaf stripe on barley. Primer pairs were designed to differentiate P. graminea from other Pyrenophora spp. using a sequence-characterized amplif ied region (SCAR) approach. A pair of P. graminea-specific primers (PG2 F/R ) was obtained that amplified a single fragment from 37 isolates of P. gram inea tested, but not from 29 isolates of other Pyrenophora spp. or 12 sapro phytes isolated from barley seed. Rapid PCR detection was achieved using a LightCycler, in which the emission of fluorescence from the binding of SYBR Green I dye to the PCR products is measured. The P. graminea-specific prod uct resulting from amplification with PG2 FIR can be distinguished from any nonspecific products by post-PCR melting point analysis. The PCR assay inv olves 40 amplification cycles of PCR, and the total PCR test including melt ing point analysis takes 25 min to complete. The rapidity of this test, com bined with the closed 'in-tube' detection of PCR products, which reduces th e potential for contamination, offers significant advantages compared with conventional laboratory and PCR analyses.