Asymmetric polymerase chain assay combined with propidium monoazide treatment and unmodified gold nanoparticles for colorimetric detection of viable emetic Bacillus cereus in milk

Abstract

Bacillus cereus is a causative agent of an emetic food-borne disease. In this study, visual detection of viable emetic B. cereus was developed using a propidium monoazide (PMA)-asymmetric polymerase chain reaction (asPCR) and unmodified gold nanoparticles (AuNPs). To detect only the viable emetic B. cereus, PMA treatment was selected before DNA extraction to eliminate the false-positive results from dead bacteria. In the presence of viable target bacteria, the long genomic DNA fragments from the cereulide synthetase gene (cesB) were produced by asPCR, which could be effectively absorbed onto naked AuNPs via coordination between Au and the nitrogen atoms of the exposed bases. After adding NaCl solution, visual detection with the naked-eye or with UV–vis spectrophotometer was possible within a few minutes. Under the optimum conditions, the limit of detection (LOD) for viable emetic B. cereus reached as low as 9.2×101 CFU/mL in 0.01M phosphate-buffered saline and 3.4×102 CFU/mL in milk, which was adequate to meet the maximum limit imposed by the Commission Regulation (EC) No 2073/2005 (500 CFU/mL). The proposed method also exhibited excellent discrimination against 10 common pathogenic bacteria in milk. The PMA-asPCR-AuNPs colorimetric assay offers a promising application in the detection of low concentrations of food-borne pathogens.