Isothermal Amplification and CRISPR/Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus

Danhong Xu; Haijuan Zeng; Wenhui Wu; Hua Liu; Jinbin Wang

doi:10.3390/foods12244432

Isothermal Amplification and CRISPR/Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus

Foods. 2023 Dec 11;12(24):4432. doi: 10.3390/foods12244432.

Authors

Danhong Xu¹, Haijuan Zeng^{2

3}, Wenhui Wu¹, Hua Liu^{2

3}, Jinbin Wang^{2

3}

Affiliations

¹ School of Food Science and Technology, Shanghai Ocean University, Shanghai 201499, China.
² Key Laboratory of Agricultural Genetics and Breeding, The Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.
³ Crops Ecological Environment Security Inspection and Supervision Center, Ministry of Agriculture and Rural Affairs, Shanghai 201106, China.

Abstract

Staphylococcus aureus exists widely in the natural environment and is one of the main food-borne pathogenic microorganisms causing human bacteremia. For safe food management, a rapid, high-specificity, sensitive method for the detection of S. aureus should be developed. In this study, a platform for detecting S. aureus (nuc gene) based on isothermal amplification (loop-mediated isothermal amplification-LAMP, recombinase polymerase amplification-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas12a) proteins system (LAMP, RPA-CRISPR/Cas12a) was proposed. In this study, the LAMP, RPA-CRISPR/Cas12a detection platform and immunochromatographic test strip (ICS) were combined to achieve a low-cost, simple and visualized detection of S. aureus. The limit of visual detection was 57.8 fg/µL of nuc DNA and 6.7 × 10² CFU/mL of bacteria. Moreover, the platform could be combined with fluorescence detection, namely LAMP, RPA-CRISPR/Cas12a-flu, to establish a rapid and highly sensitive method for the detection of S. aureus. The limit of fluorescence detection was 5.78 fg/µL of genomic DNA and 67 CFU/mL of S. aureus. In addition, this detection platform can detect S. aureus in dairy products, and the detection time was ~40 min. Consequently, the isothermal amplification CRISPR/Cas12a platform is a useful tool for the rapid and sensitive detection of S. aureus in food.

Keywords: LAMP; RPA; S. aureus; fluorescence detection; immunochromatographic test strip.

Grants and funding

This work was supported by the SciTech Funding by CSPFTZ Lingang Special Area Marine Biomedical Innovation Platform, Shanghai Frontiers Research Center of the Hadal Biosphere, Shanghai Agricultural Science and Technology Innovation Action Plan (21N31900800), Project supported by the National Natural Science Foundation of China (Grant No. 82173731).