ABSTRACT
@#Aims: Comparison between ZEN™ double-quenched probe and SYBR GreenER™ real-time PCR assay to develop asensitive 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. Theperformance of ZEN™ double-quenched probe and SYBR GreenER™ chemistry were compared by using knownconcentrations of purified DNA. ZEN™ double-quenched probe showed a dynamic range of 3 log units and sensitivity of600 fg/reaction or 100 copies/reaction. SYBR GreenER™ chemistry had a wider quantitative dynamic range of 6 logunits 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 forSYBR GreenER™ assay and ZEN™ probe chemistry in DNA extracted directly from artificially inoculated milk, with thelowest limit of detection by SYBR GreenER™ assay in the range of 6 fg/reaction or 25 copies/mL and it quantifiedBacillus cereus in milk with high relative accuracy.Conclusion: SYBR GreenER™ assay provides a fast, sensitive and specific detection and quantification ofenterotoxigenic Bacillus cereus and allowed a direct assessment and quantification of Bacillus cereus from milk foodsample.Conclusion, significance and impact of study: The study shows an efficient, specific and highly sensitive method ofdirectly assessing the enterotoxigenic Bacillus cereus from milk product, using cheaper dsDNA binding SYBRGreenER™ dye.
ABSTRACT
Aims: The present study aimed to develop a new approach for detecting Salmonella species at picogram levels using magnetic bead (MB) aggregation through loop-mediated isothermal amplification (LAMP). Methodology and results: For the first time to our knowledge, Salmonella LAMP amplicons were analyzed using MB aggregation. LAMPs were conducted with a simple heat block, and the results were compared with those obtained with conventional LAMP-MB techniques. Furthermore, the volume and concentration of MB solutions were optimized. Our method detected Salmonella genomic DNA at a low picogram level (1 pg/µL). The specificity of this method was also examined using other bacterial species. Owing to specific Salmonella primers, the use of LAMPs approach was time effective; because these amplicons could be utilized after 20 min instead of the 1 h needed for conventional methods. Furthermore, LAMP-positive amplicons were rapidly detected within 5 min. Conclusion, significance and impact study: The determination of DNA in biological samples is a recent keystone in genomic analysis techniques. Salmonella is a foodborne pathogen that causes many diseases and, in extreme cases, death. Accordingly, detecting Salmonella has become a vital issue for food safety and security. Combining DNA and MBs on paper helped us to develop a new method for label-free, non-immobilized, naked eye detection of Salmonella. The process is very specific owing to the use of exact primers and does not require heavy or expensive instrumentation. In the future, this method could be applied to biosensors as well as in biomedical and molecular diagnostic fields.