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Objective To develop a rapid and visual method for detecting Shigella by combining loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats and associated protein 12b (CRISPR/Cas12b). Methods Based on the conserved invasion plasmid antigen H gene ipaH7 sequence of Shigella, LAMP primers were designed and screened to establish a LAMP method for detection of Shigella. The candidate sgRNAs were designed based on the target DNA sequence of LAMP amplified fragment, and then the sgRNA, which could specifically recognize and stimulate Cas12b cleavage, was screened. Finally, a rapid detection method for detecting Shigella was developed by combining LAMP with CRISPR/Cas12b, and the specificity and sensitivity were evaluated. Results A set of LAMP primers and sgRNA targeting Shigella ipaH7 were screened, and a LAMP CRISPR/Cas12b detection method was established. The detection could be completed within 1 hour with the lowest limit of detection of 1.1×101 CFU/mL for pure culture and 1.1×101 CFU/g for spiked pork samples, and no cross reactivity with other pathogens. Conclusion This study successfully establishes a rapid, sensitive and visual detection method for Shigella, which can achieve accurate detecting Shigella in food samples.
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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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