Rapid and sensitive detection of Pectobacterium carotovorum subsp. carotovorum in fresh produce using silica-coated magnetic nanoparticles combined with filtration-based real-time PCR

Pectobacterium carotovorum subsp. carotovorum (Pcc) possesses a powerful enzymatic mechanism that destroys plant-cell walls. Therefore, Pcc is the etiology of soft-rot disease in several crops and is recognized as one of the most important pathogens in agriculture. Soft-rot is difficult to control at the source when infection occurs. Thus, it is essential to develop sensitivity monitoring and diagnostic methods for timely control measures. Molecular detection methods, such as real-time polymerase chain reaction (PCR), are widely used for detecting Pcc. However, because Pcc is present at low levels in initial soft-rot infection, enrichment and efficient pathogen-separation methods are needed for sensitive monitoring. In this study, filtration was used as a pretreatment method for pathogen concentration, and silica-coated magnetic nanoparticles (SMN)- based real-time PCR was employed to detect low levels of Pcc in fresh produce. As a result, 102 CFU/10 mL was detected by various DNA-extraction methods in pure culture, whereas SMN detected up to 101 CFU/10 mL. In addition, when DNA was extracted using SMN after filtration using a cellulose acetate filter, the sensitivity of real-time PCR was improved to 10° CFU/10 mL. In conclusion, the pretreatment method including SMN, and concentration procedures increased the detection sensitivity by 100-fold; in potato and chicory, 101 CFU/g was detected at a frequency of 90% of the samples, and 10° CFU/g was detected at 70%; and in Kimchi Cabbage, 101 CFU/g was detected at 100% and 10° CFU/g was detected at 80%.