A quantitative PCR based method using propidium monoazide for specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum in kimchi cabbage (Brassica rapa L. subsp. pekinensis)

Abstract

Quality issues related to bacterial soft rot caused by Pectobacterium carotovorum ssp. carotovorum (PCC) in commercially available kimchi highlights the demand for rapid, specific, sensitive, and accurate methods for monitoring the quality of kimchi cabbage. Moreover, development and optimization of analytical methods to determine and quantify only the viable bacterial cells and not the non-viable cells from cabbage samples are indispensable. In this study, a quantitative PCR (RT-qPCR) method combined with an intercalating propidium monoazide (PMA) dye used as the DNA-binding molecule was evaluated for selective detection of viable PCC cells in bacterial suspension of live and dead cells and in artificially inoculated fresh and kimchi cabbage samples. The recovery rates of PCC cells using RT-qPCR from fresh and kimchi cabbage test samples, artificially inoculated with approximately 4.76 log10 DNA genomic copies/reaction, were 94.1 ± 6.2% and 96.80 ± 5.1%, respectively. The optimum concentration of PMA was 16 μM. The quantification data obtained in the PMA/RT-qPCR assays showed that CT values were significantly increased with the decreasing proportion of viable cells in bacterial suspension and cabbage samples. The PMA/RT-qPCR assay presented herein could selectively detect viable PCC cells and be useful for monitoring the quality of kimchi cabbage.