A multiplex PCR method for detection of Clavibacter michiganensis subsp. michiganensis with co-amplification of its host DNA

Abstract

A multiplex PCR assay system was developed for the detection of Clavibacter michiganensis subsp. michiganensis (Cmm), which combined two tests in one reaction mixture. Cmm-specific primers PSA-4/PSA-R and Solanum lycopersicum–specific primers NS-7-F/NS-8-R (internal PCR control primer) were combined in one PCR reaction mixture with Cmm and plant DNA as template. The primer sets could amplify the target product successfully. Different combinations and concentrations of primers and annealing temperatures were tested, respectively. The detection level of the optimized multiplex PCR assay was up to 5×10² cfu·mL^-1. To verify the applicability of this system, it was employed to detect Cmm in tomato seeds and plantlet samples. Seeds mixed with Cmm and diseased plantlets were detected successfully. The multiplex PCR system will avoid false-negative results and provide a reliable method for the detection of Cmm.

Keywords

Clavibacter michiganensis subsp. michiganensis (Cmm) / molecular detection / multiplex PCR / internal PCR control / false negative

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Acknowledgements

This research was financially supported by the doctor’s foundation of Hebei Province, China (No. 05547005D-1).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg