A comparison of ZEN double-quenched probe and SYBR GreenER chemistries in the real-time PCR based quantitative detection of enterotoxigenic Bacillus cereus in milk

Aims: Comparison between ZEN™ double-quenched probe and SYBR GreenER™ real-time PCR assay to develop a sensitive and specific assay for the direct detection and quantification of enterotoxigenic Bacillus cereus in milk.Methodology and results: Novel primers and probe were designed to target the enterotoxigenic nhe gene.The performance of ZEN™ double-quenched probe and SYBR GreenER™ chemistry were compared by using known concentrations of purified DNA.ZEN™ double-quenched probe showed a dynamic range of 3 log units and sensitivity of 600 fg/reaction or 100 copies/reaction.SYBR GreenER™ chemistry had a wider quantitative dynamic range of 6 log units with sensitivity down to 6 fg/reaction or 1 copy number/reaction.Thus, SYBR GreenER™ chemistry was 100× more sensitive with wider quantification range compared to ZEN™ probe chemistry.Similar result was also found for SYBR GreenER™ assay and ZEN™ probe chemistry in DNA extracted directly from artificially inoculated milk, with the lowest limit of detection by SYBR GreenER™ assay in the range of 6 fg/reaction or 25 copies/mL and it quantified Bacillus cereus in milk with high relative accuracy.Conclusion: SYBR GreenER™ assay provides a fast, sensitive and specific detection and quantification of enterotoxigenic Bacillus cereus and allowed a direct assessment and quantification of Bacillus cereus from milk food sample. Conclusion, significance and impact of study:The study shows an efficient, specific and highly sensitive method of directly assessing the enterotoxigenic Bacillus cereus from milk product, using cheaper dsDNA binding SYBR GreenER™ dye.