跨越式滚环等温扩增技术结合CRISPR/Cas12a定量检测海产品中的副溶血性弧菌

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

将CRISPR/Cas12a系统与跨越式滚环等温扩增技术(saltatory rolling circle amplification,SRCA)相结合,建立一种快速、灵敏定量检测海产品中副溶血性弧菌(Vibrio parahemolyticus)的方法(SRCA-Cas12a)。通过SRCA扩增靶标DNA,CRISPR/Cas12a系统识别靶标DNA并裂解单链报告探针,从而建立SRCA-Cas12a检测方法。分析该方法的特异性与灵敏度,并进行加标回收实验和实际样品检测。结果显示该方法可区分副溶血性弧菌和其他食源性致病菌,特异性良好。在最优检测体系下,建立了副溶血性弧菌菌液浓度的对数与荧光信号强度的线性关系,线性方程为y=1.8472x+2.4738(R^(2)=0.9866),检出限为3.6 CFU/mL(R_(SN)=3)。在人工污染样品中副溶血性弧菌的加标回收率为95.5%~104.4%。在实际样品检测中,该方法的敏感性为100.0%、特异性为95.2%、符合率为97.2%。本研究所建立的SRCA-Cas12a方法具有特异性好、检测限低的优点,为副溶血性弧菌的快速定量检测提供了一种新策略。

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=1.8472x+2.4738(R^(2)=0.9866)and the detection limit was 3.6 CFU/mL(R_(SN)=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.