Abstract:

This study aimed to establish a real-time fluorescence loop-mediated isothermal amplification assay to detect
Bacillus cereus. Based on the published Bacillus cereus hblA gene sequence, primers were designed, and the intercalating
dye SYBR-Green I was added to the reaction system, which allowed the reaction products to be measured by ESE-Quant
Tube Scanner. The products were identified by restriction digestion followed by electrophoresis. The performance of the
assay was evaluated with respect to specificity and sensitivity in comparison with the ordinary LAMP, and the limit of
detection (LOD) for artificially contaminated milk was tested. Among 21 strains tested, four Bacillus cereus strains were
identified, the other 17 strains were negative. The sensitivity for Bacillus cereus was 8.2 CFU/mL in pure cultures, which
was 10 times more sensitive than the ordinary LAMP. The LOD for artificially contaminated milk was 8.2 CFU/mL. The
result could be obtained in 20 minutes. This method has high specificity and sensitivity and is less time-consuming, requiring
only simple equipment and allowing real-time monitoring. It is expected to be applied as an effective method for rapid
detection of B. cereus.

Key words: real-time fluorescence loop-mediated isothermal amplification (RealAmp), Bacillus cereus, detection, milk