Abstract:

【Objective】 Reaction components and parameters of triplex PCR system were optimized to establish the method for the detection of Botrytis cinerea, Colletotrichum gloeosporioides and Verticillium dahliae simultaneously at early stage. 【Method】Three sets of specific primers were selected and the annealing temperature of PCR reaction was optimized. The three levels of six factors (three primers, Taq DNA polymerase, dNTP and Mg2+) in multiplex PCR system were evaluated using orthogonal design method. 【Result】 The triplex PCR system for simultaneous detection of these three pathogens in strawberry was successfully established and verified. The volume of reaction system which could amplify three specific bands of 729 bp, 539 bp and 450 bp was 25 μL and the annealing temperature was 50℃. The reaction mix contained 0.32 μmol?L-1 of primer C729+/-, 0.032 μmol?L-1 of primer DB19/DB2, 0.32 μmol?L-1 of primer CgInt/ITS4, 1.5 U Taq DNA polymerase, 0.15 mmol?L-1 dNTP and 1.6 mmol?L-1 MgCl2. 【Conclusion】 The triplex PCR system can detect B. cinerea, C. gloeosporioides and V. dahliae in infected plant tissues and soil with the sensitivity of 10 pg DNA.

Key words: Strawberry')">Strawberry, Botrytis cinerea, Colletotrichum gloeosporioides, Verticillium dahliae, molecular detection, triplex PCR