DNase I treated DNA-PCR based detection of food pathogens immobilized by metal hydroxides

Abstract

Bacterial immobilization by metal hydroxides can be used for enrichment of various bacterial strains including Gram (+) and Gram (−). The polymerase chain reaction (PCR)-based bacterial detection without enrichment culture could be implemented by concentrating bacteria from food matrix by metal hydroxides. To distinguish between viable and non-viable cells, it is often required to detect the mRNA, an indicator of viable cells. This technique, although provides accurate and reliable result, is expensive and time-consuming. Here, we report the studies on application of DNase I treatment to eliminate DNA from dead cells and subsequently detect the presence of viable pathogens by conventional PCR. It was found that treatment of immobilized cells with DNase I for 1 h at 37°C prior to DNA extraction could efficiently eliminate false positives due to the presence of non-viable cells. The technique was used to detect the presence of various pathogens in food model. The detection limits for Escherichia coli O157:H7 (384 bp), Listeria monocytogenes (482 bp), and E. coli wild type (580 bp) was 5 × 10¹ cells and that for Salmonella typhimurium (685 bp) was 5 × 10² cells in 10 ml of whole milk.