Establishment of LAMP combined with CRISPR/Cas12a system for detecting tlh gene of Vibrio parahaemolyticus and its evaluation
10.13481/j.1671-587X.20250529
- VernacularTitle:LAMP联合CRISPR/Cas12a系统检测副溶血性弧菌tlh基因方法的建立及其评价
- Author:
Yujiao ZHOU
1
;
Jifei YANG
;
Yan LIU
;
Wenbo DING
;
Xianyu ZHANG
;
Jianyu YANG
;
Linran GAO
;
Yundong ZHAO
;
Liyuan SUN
Author Information
1. 北华大学医学技术学院临床病原学检验教研室,吉林吉林 132013
- Keywords:
Vibrio parahaemolyticus;
CRISPR-Cas12a system;
tlh gene;
Loop-mediated isothermal amplification;
Nucleic acid test
- From:
Journal of Jilin University(Medicine Edition)
2025;51(5):1399-1406
- CountryChina
- Language:Chinese
-
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.
