Multiplex polymerase chain reaction detection of Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected maize leaf tissues

Abstract

Multiplex polymerase chain reaction (PCR) is a method for simultaneous identification and detection of multiple pathogenic fungi, however, its complexity limits its application. To simplify the protocol and to improve the effectiveness, three-level designs for six factors (three primers, Taq DNA polymerase, dNTP, Mg2+ ) were constructed to optimize the multiplex PCR system by using the orthogonal design method and the annealing temperature of the PCR reactions was also optimized. Finally, a multiplex PCR system for the simultaneous detection of these three pathogens of maize was successfully established. The reaction volume was 25 μl and the annealing temperature was 57℃. The optimal conditions for multiplex PCR reaction contained 0.48 μmol/L Cl-1/Cl-2, 0.72 μmol/L Bm-1/Bm-2, 0.24 μmol/L Az-1/Az-2, 1.5 U polymerase, 0.35 mmol/L dNTP, and 1.25 mmol/L MgCl2 . The multiplex PCR system can detect Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected plant tissues rapidly with the sensitivity at 10 pg DNA/μl.