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MULTIPLEX-TOUCHDOWN PCR FOR RAPID SIMULTANEOUS DETECTION OF Rhizoctonia cerealis AND Rhizoctonia solani О. Hrytsev, J. Shevchenko, N. Vorobiova, L. Skivka

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"Biotechnologia Acta" V. 12, No 3, 2019
https://doi.org/10.15407/biotech12.03.075

MULTIPLEX-TOUCHDOWN PCR FOR RAPID SIMULTANEOUS DETECTION OF Rhizoctonia cerealis AND Rhizoctonia solani

О. Hrytsev, J. Shevchenko, N. Vorobiova, L. Skivka

Abstract

The aim of the study was to develop rapid and sensitive assay for the simultaneous detection of R. cerealis and R. solani. Pure cultures of fungi were grown on a potato dextrose agar for 5 days at 28 °C, and mycelium was harvested and used for DNA extraction. Total DNA was extracted using a commercial test-systems. Molecular identification of phytopathogenic fungi was performed using a multiplex-touchdown PCR with further electrophoretic separation of amplification products in agarose gel. The specific sequence characterized amplified region primers RtubR4/RtubF4 for R. cerealis and ITS1/GMRS–3 for R. solani were tested for their specificity and useability in PCR multiplex capacity. The specificity of the multiplex-touchdown PCR was tested using DNA from wide range of fungal species and non-target DNA from healthy wheat. The used primer pairs provided only specific fragments for R. cerealis and R.solani. No PCR products were obtained during amplification with the negative control or non-target DNA templates from other species. Coupled to this we have optimized the temperature regime for the multiplex PCR protocol. Taken together, our protocol convincingly demonstrated the simultaneous ability to detect Rhizoctonia cerealis and Rhizoctonia solani and can be used for the diagnosis of compound Rhizoctonia root rot.

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