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

打开文本图片集

[中图分类号] R446.5 [文献标志码] A

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（Casl2a）（CRISPR-Casl2a） system，and to evaluate itsefficacy for detecting the thermolabile hemolysin （tlh） gene of Vibrio parahaemolyticus （Vρ） .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. Bacilus 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/ Casl2a method for detecting the tlh gene of Vϕ .Results：The method specifically detected Vϕ ，while Bacillus cereus， Staphylococcus aureus，and Escherichia coli yielded negative results. The DNA extraction concentration of Vϕ was 1 90.67mg⋅L-1 with an A（26O）/（A28O） ratio of 1.84. Under the reaction conditions of 37Y with 80 cycles for 40min using quantitative PCR（qPCR）method，when the concentrations of Casl2a protein and crRNA in the LAMP-CRISPR/Casl2a system were 50nmol⋅L-1 ，the visual brightness and relative fluorescence intensity peaks were high. The sensitivity of LAMP CRISPR/Casl2a for detecting （204号 Vp DNA concentration could reach 10-6mg⋅L-1 . The reproducibility test results showed that different experimenters had consistent results in different experimental environments and times. Conclusion： The established LAMP-CRISPR/Casl2a method can rapidly detect the tlh gene of VP with high sensitivity and specificity，and can achieve short-term visual detection in the field.

KEYWORDsVibrio parahaemolyticus； CRISPR-Casl2a system； tlh gene；Loop-mediated isothermalamplification；Nucleic acid test

副溶血性弧菌（Vibrioparahaemolyticus， VP ）是一种常见的食源性病原体，主要存在于海产品和盐渍食品[1；该菌可引发食物中毒并导致急性肠胃炎，患者病情发展迅速，不及时诊断干预有致死风险[2-3]。（剩余9000字）