基于CRISPR/Cas12a的室温稳定鼠疫耶尔森氏菌检测体系的建立

Establishment of an ambient temperature stable CRISPR/Cas12a nucleic acid detection system for Yersinia pestis

目的筛选并优化冻干保护剂组合,建立可室温稳定储存的、不影响试剂检测灵敏度的Cas12a鼠疫菌核酸检测冻干体系,为其应用于动物鼠疫监测的现场快速检测奠定基础。方法以鼠疫耶尔森氏菌pla基因为靶基因,选择9种常用冻干保护剂加入核酸检测试剂中,筛选有明显保护效果的4种保护剂进行优化。按照正交实验设计原则设计保护剂组合,通过37℃加速破坏实验及26℃室温稳定性实验对复合保护剂进行保护效果和灵敏度评价。结果加入蔗糖、山梨醇和海藻糖3种保护剂的冻干检测体系可在37℃稳定存放2周。室温下存放3个月后体系检测信号强度与37℃加速破坏1周时基本相当,同时检测方法的灵敏度依然能达到10-5 ng/μL,说明Cas12a检测试剂加入筛选得到的保护剂冻干后,可在室温下稳定保存至少6个月。结论本研究构建的室温稳定CRISPR/Cas12a鼠疫菌检测体系可在室温下稳定存放至少半年,能够满足鼠疫菌现场快速检测中对试剂稳定性的需求。同时,本研究获得的复合保护剂组分对构建其它室温稳定CRISPR/Cas12a核酸检测体系具有重要参考价值。

The instability of reagents for CRISPR/Cas12a-based nucleic acid detection techniques at room temperature strongly inhibits their application in point-of-care testing.This study was aimed at screening lyophilized protective agents,optimizing the lyoprotectant composition and establishing a Cas12a-based detection system that retains high detection sensitivity after storage at room temperature.Using the pla gene of Yersinia pestis as the target gene for detection,we evaluated the efficacy of nine common protectants in the Cas12a-based nucleic acid detection system.Four protectants with significant protective effects were selected for further optimization.A variety of protectant combinations were optimized through an orthogonal experiment design,and their protective efficacy and detection sensitivity were evaluated in accelerated shelf-life tests at 37℃and stability experiments at 26℃.The Cas12a-based nucleic acid detection reagents supplemented with a combination of protectants containing sucrose,sorbitol and trehalose were stably stored at 37℃for 2 weeks without a significant loss of detection sensitivity or decrease in detection signal.After storage at 26℃for 3 months,the same lyophilized detection agent achieved detection results similar to those after storage at 37℃for 1 week,thus suggesting that the reagent remained stable at 26℃for at least 6 months.The ambient temperature stable CRISPR/Cas12a nucleic acid detection system for Y.pestis established herein can be stored at room temperature for at least half a year,thus meeting reagent stability requirements for in-field detection in animal plague surveillance.The composition of the lyoprotectants optimized in this study provides valuable information for the construction of various other ambient temperature stable CRISPR/Cas12a-based nucleic acid detection methods for point-of-care testing.