Abstract:

Objective To establish a rapid detection method for pathogenic microorganisms by combining loop-mediated isothermal amplification （LAMP） and clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 12a（Cas12a） （CRISPR-Cas12a） system， and to evaluate its efficacy for detecting the thermolabile hemolysin （tlh） gene of Vibrio parahaemolyticus（Vp）. Methods Using the tlh gene of Vp as the target gene， LAMP primers and CRISPR RNA（crRNA） were designed to construct and optimize the optimal concentration ratio of each component in the LAMP-CRISPR detection system. Bacillus cereus， Staphylococcus aureus， and Escherichia coli were used as control groups， and the specificity， sensitivity， reproducibility and positive conformity rate were verified to establish a rapid LAMP-CRISPR/Cas12a method for detecting the tlh gene of Vp. Results The method specifically detected Vp， while Bacillus cereus， Staphylococcus aureus， and Escherichia coli yielded negative results. The DNA extraction concentration of Vp was 190.67 mg·L^-1 with an A（260）/（A280） ratio of 1.84. Under the reaction conditions of 37 ℃ with 80 cycles for 40 min using quantitative PCR （qPCR） method， when the concentrations of Cas12a protein and crRNA in the LAMP-CRISPR/Cas12a system were 50 nmol·L^-1， the visual brightness and relative fluorescence intensity peaks were high. The sensitivity of LAMP CRISPR/Cas12a for detecting Vp DNA concentration could reach 10^-6 mg·L^-1. The reproducibility test results showed that different experimenters had consistent results in different experimental environments and times. Conclusion The established LAMP-CRISPR/Cas12a method can rapidly detect the tlh gene of Vp with high sensitivity and specificity， and can achieve short-term visual detection in the field.

Key words: Vibrio parahaemolyticus, CRISPR-Cas12a system, tlh gene, Loop-mediated isothermal amplification, Nucleic acid test