FOOD SCIENCE ›› 2022, Vol. 43 ›› Issue (14): 289-295.doi: 10.7506/spkx1002-6630-20210917-218

• Safety Detection • Previous Articles    

Saltatory Rolling Circle Amplification Combined with CRISPR/Cas12a for Quantitative Detection Vibrio parahaemolyticus in Seafoods

DONG Huanzhe, YUAN Ning, ZHANG Yunzhe, YANG Qian, LU Xin, GUO Wei, ZHANG Wei   

  1. (1. College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China; 2. College of Science and Technology, Hebei Agricultural University, Cangzhou 061100, China; 3. College of Public Health, Hebei University, Baoding 071002, China; 4. College of Life Sciences, Hebei Agricultural University, Baoding 071001, China; 5. Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganism, Baoding 071001, China)
  • Published:2022-07-28

Abstract: This study aimed to develop a rapid and sensitive method for the quantitation of Vibrio parahemolyticus in seafoods by combining the CRISPR/Cas12a system with saltatory rolling circle amplification (SRCA-Cas12a). The target DNA of V. parahaemolyticus was amplified by SRCA and recognized by the CRISPR/Cas12a system and the single-stranded reporter probe was cleaved by the CRISPR/Cas12a system. The sensitivity, specificity and spiked recovery of SRCA-Cas12a were tested. The results indicated that SRCA-Cas12a could distinguish V. parahaemolyticus from other food-borne pathogens with excellent specificity. Under optimal conditions, a linear relationship between the logarithm of the concentration of V. parahaemolyticus and the intensity of fluorescence signal was established, which was fitted as follows: y = 1.847 2x + 2.473 8 (R2 = 0.986 6) and the detection limit was 3.6 CFU/mL (RSN = 3). The spiked recoveries of V. parahaemolyticus in artificially contaminated samples were 95.5%–104.4%. The method was applied for the detection of actual samples with a sensitivity of 100.0%, a specificity of 95.2%, and a coincidence rate of 97.2%. Hence, this developed method has the advantages of excellent specificity and low detection limit, thereby providing a new strategy for rapid quantitative detection of V. parahaemolyticus.

Key words: CRISPR/Cas12a; saltatory rolling circle amplification; Vibrio parahemolyticus; quantitative detection; seafood

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