Full
Outline
To establish a dual recombinase aided amplification (RAA) and clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) technology for the rapid detection of Bacillus cereus and Listeria monocytogenes.
Using the gyrB gene and the listeriolysin virulence gene hly as detection targets, RAA primers and crRNAs were designed. Their sensitivity and specificity were analyzed and the positive detection rate of this method was evaluated using actual samples.
The dual RAA/CRISPR-Cas12a technology could detect 0.0001 ng/μL of Bacillus cereus genomic DNA and 0.001 ng/μL of Listeria monocytogenes genomic DNA within 40 minutes at 37 ℃, with no cross-reactivity against other foodborne pathogens (Salmonella spp., Staphylococcus aureus, Escherichia coli, Clostridium perfringens, Vibrio parahaemolyticus). Using real-time polymerase chain reaction as a reference, 30 beef samples were tested. The RAA/CRISPR-Cas12a method detected 5 positive samples, achieving a positive detection rate of 100%, consistent with the real-time polymerase chain reaction method.
The dual RAA/CRISPR-Cas12a detection method established in this study offers rapid detection, high sensitivity, and high specificity, making it suitable for detecting Bacillus cereus and Listeria monocytogenes in food.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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