Fast EvaGreen Real-time Duplex PCR for the Individual Detection of Staphylococcus aureus and Bacillus cereus using a Uniform Amplification Strategy

Abstract

: Reliable and sensitive detection of Bacillus cereus ( B. cereus ) and Staphylococcus aureus ( S. aureus ) is needed to limit the outbreak of food poisoning thereof. This paper reports the development of two individual duplex real-time PCR assays with subsequent melting curve analyses based on EvaGreen ® dye for dual gene detections of two bacteria under uniform amplification condition . The duplex assays targeted thermostable nuclease gene ( nuc ) and heat-shock protein gene ( htrA ) of S. aureus and non-haemolytic enterotoxin gene ( nhe ) and cereolysin A gene ( cerA ) for B. cereus detection. The assays successfully detected both the species with high specificity and sensitivity in genomic DNA samples and in simulated real milk samples. The selectivity was also confirmed against a wide range of background microflora. Sensitivity of 500 cell/mL and 25 cell/mL of milk was obtained respectively for S. aureus and B. cereus . The proposed methodology allowed for fast, inexpensive, selective and sensitive multi-targets detections of both bacteria in a single amplification run on multiple genes to detect S. aureus and B. cereus in milk product by using dsDNA binding EvaGreen ® dye.