RPA-Cas12a-FS: A frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification

Hua Liu; Jinbin Wang; Haijuan Zeng; Xiaofeng Liu; Wei Jiang; Yu Wang; Wanbao Ouyang; Xueming Tang

doi:10.1016/j.foodchem.2020.127608

RPA-Cas12a-FS: A frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification

Food Chem. 2021 Jan 1:334:127608. doi: 10.1016/j.foodchem.2020.127608. Epub 2020 Jul 19.

Authors

Hua Liu¹, Jinbin Wang², Haijuan Zeng¹, Xiaofeng Liu³, Wei Jiang¹, Yu Wang³, Wanbao Ouyang³, Xueming Tang⁴

Affiliations

¹ Institute of Biotechnology Research, Shanghai Academy of Agricultural Sciences; Key Laboratory of Agricultural Genetics and Breeding, 2901 Beidi Road, Shanghai 201106, China; Crops Ecological Environment Security Inspection and Supervision Center (Shanghai), Ministry of Agriculture and Rural Affairs, P.R.C., 2901 Beidi Road, Shanghai 201106, China.
² Institute of Biotechnology Research, Shanghai Academy of Agricultural Sciences; Key Laboratory of Agricultural Genetics and Breeding, 2901 Beidi Road, Shanghai 201106, China; Crops Ecological Environment Security Inspection and Supervision Center (Shanghai), Ministry of Agriculture and Rural Affairs, P.R.C., 2901 Beidi Road, Shanghai 201106, China. Electronic address: [email protected].
³ School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China.
⁴ Institute of Biotechnology Research, Shanghai Academy of Agricultural Sciences; Key Laboratory of Agricultural Genetics and Breeding, 2901 Beidi Road, Shanghai 201106, China; Crops Ecological Environment Security Inspection and Supervision Center (Shanghai), Ministry of Agriculture and Rural Affairs, P.R.C., 2901 Beidi Road, Shanghai 201106, China. Electronic address: [email protected].

PMID: 32711280
DOI: 10.1016/j.foodchem.2020.127608

Abstract

Food analysis to ensure food safety and quality are relevant to all countries. This study aimed to develop a detection technique by combining recombinase polymerase amplification with CRISPR-Cas12a for food safety (termed RPA-Cas12a-FS). Our data showed that this novel method could be detected via fluorescence intensity for the molecular identification of foodborne pathogenic bacteria, genetically modified crops, and meat adulteration. After optimization, the sensitivity and stability of RPA-Cas12a-FS was further enhanced. The RPA-Cas12a-FS system could specifically detect target gene levels as low as 10 copies in 45 min at 37 °C. The RPA-Cas12a-FS system was sensitive both using standard samples in the lab and using samples from the field, which indicated that this detection method was practical. In conclusion, a simple, rapid, and highly sensitive detection method based on CRISPR-Cas12a was developed for molecular identification in the food safety field without requiring technical expertise or ancillary equipment.

Keywords: CRISPR-Cas12a; Detection; Foodborne pathogenic bacteria; Genetically modified crops; Meat adulteration.

MeSH terms

Bacterial Proteins / genetics
CRISPR-Associated Proteins / genetics
CRISPR-Cas Systems*
Crops, Agricultural / genetics
Endodeoxyribonucleases / genetics
Fluorescence
Food Analysis / methods*
Food Contamination / analysis*
Food Microbiology / methods*
Food Safety
Meat
Nucleic Acid Amplification Techniques / methods*
Plants, Genetically Modified / genetics
RNA, Guide, CRISPR-Cas Systems
Recombinases / genetics
Sensitivity and Specificity

Substances

Bacterial Proteins
CRISPR-Associated Proteins
RNA, Guide, CRISPR-Cas Systems
Recombinases
Cas12a protein
Endodeoxyribonucleases