基于CRISPR/Cas13a-HCR的副溶血性弧菌荧光检测方法构建及应用

Development and application of a fluorescence method for Vibrio parahaemolyticus detection based on CRISPR/Cas13a and hybridization chain reaction

摘要:

目的 将CRISPR系统与杂交链式反应(hybridization chain reaction, HCR)结合，构建一种用于副溶血性弧菌快速检测的荧光方法，以实现对病原菌的快速、灵敏、准确检测。方法 筛选副溶血性弧菌特异性保守序列，设计级联探针(RP/I)及HCR发夹结构，优化反应体系及实验条件，评估该方法的可行性、灵敏度和特异性，并利用副溶血性弧菌污染的水产品验证其抗干扰能力。结果 仅当靶标存在时，CRISPR/Cas13a才能被激活，反式切割RP/I级联探针，释放出I链并引发HCR反应，产生明显的荧光增强信号。该方法具有良好的检测特异性，可区分单碱基错配、双碱基错配和三碱基错配，并能针对不同菌株有效区分副溶血性弧菌与非靶标菌(如溶藻弧菌、创伤弧菌、哈维氏弧菌、霍乱弧菌和大肠杆菌)。基于纯靶标RNA的检测灵敏度为1.01 pmol/L，在25 pmol/L-10 nmol/L具有良好的线性关系，其回归方程为y=7 236.75×lg C_T-RNA-8 590.11，R²=0.99。本研究方法可快速检测多种水产品中副溶血性弧菌提取的RNA，检测结果与实时荧光定量逆转录PCR (real-time reverse transcription quantitative PCR, RT-qPCR)一致。结论 本研究建立的CRISPR/Cas13a-HCR荧光检测方法可快速、准确地检测副溶血性弧菌，且具有良好的灵敏度、特异性和准确性。

Abstract:

Objective To develop a fluorescence method for Vibrio parahaemolyticus detection by the combination of CRISPR system and the hybridization chain reaction (HCR), thus achieving rapid, sensitive, and accurate detection of the pathogen.Methods Cascade probe (RP/I) and HCR hairpin structures were first designed according to a specific conserved sequences screened from V. parahaemolyticus. Subsequently, the feasibility, specificity, and sensitivity of the method were evaluated after the optimization of reaction conditions. Furthermore, V. parahaemolyticus-contaminated aquatic products were used to validate the interference resistance of the method.Results The cleavage of CRISPR/Cas13a was activated upon binding to the target RNA (T-RNA), leading to the trans-cleavage of the RP/I cascade probe and the release of I strand. Then, the released I strand subsequently triggered HCR, generating a significant fluorescence signal for target detection. The established method successfully distinguished target sequences with single-base, double-base, and triple-base mismatches and enabled the specific identification of V. parahaemolyticus against other non-target bacteria, including V. alginolyticus, V. vulnificus, V. harveyi, V. cholerae, and Escherichia coli, demonstrating excellent specificity. The assay showed a good linear correlation over a T-RNA concentration range of 25 pmol/L to 10 nmol/L. The corresponding linear regression equation was y=7 236.75×lg C_T-RNA-8 590.11 (R²=0.99, C represents the T-RNA concentration and y represents the fluorescence intensity), with the LOD of 1.01 pmol/L. The proposed method enabled rapid detection of RNA extracted from V. parahaemolyticus in various aquatic products, yielding results consistent with those obtained by RT-qPCR.Conclusion The fluorescence method based on CRISPR/Cas13a-HCR established in this study realizes rapid detection of V. parahaemolyticus, demonstrating good sensitivity, specificity, and accuracy.