Super-Fast Detection of Bacillus cereus by Combining Cellulose Filter Paper-Based DNA Extraction, Multienzyme Isothermal Rapid Amplification, and Lateral Flow Dipstick (MIRA-LFD).

Abstract

Bacillus cereus is a widespread foodborne pathogen that can cause food poisoning when present in food at certain levels. Ingesting contaminated food may lead to symptoms such as abdominal pain, diarrhea, and, in severe cases, life-threatening conditions. In this study, a simple and super-fast method for detecting B. cereus was developed, which combines cellulose filter paper-based DNA extraction, multienzyme isothermal rapid amplification (MIRA), and lateral flow dipstick (LFD) technology. Initially, PCR was adopted to evaluate the DNA extraction efficiency of the filter paper, followed by the optimization of the lysis formula and extraction conditions. With the above optimization, DNA that can be used for subsequent nucleic acid amplification can be obtained within 3 min. Then, the isothermal amplification of MIRA-LFD was established and optimized to evaluate the detection specificity and sensitivity. Finally, the developed method was applied to detect B. cereus in cooked rice samples. The results indicated that the entire amplification procedure of MIRA-LFD only takes 15 min at 39 °C. The whole super-fast detection system could be completed in less than 20 min, from DNA extraction to result interpretation, which achieved a detection limit of 12 fg/μL of DNA concentration, corresponding to approximately 115 CFU/mL in actual samples.