基于CRISPR/Cas12a的核酸便捷化检测方法和现场快速便携式检测装置

Convenient Nucleic Acid Detection Method and Point-of-Care Detection Device Based on CRISPR/Cas12a Molecular Diagnosis

核酸检测是病原体检测的必备流程,也是提升重大疾病和传染病应对能力、实施精准医疗的关键,在疾病防控、临床诊断、生物安全、环境监测等领域被广泛应用。目前临床上常用实时荧光定量聚合酶链式反应(PCR)方法进行核酸检测,但该方法的检测周期较长,且对实验室环境、硬件设备等的要求较高。本文提出了一种基于CRISPR/Cas12a(Cpf1)的核酸便捷化检测方法,并设计了一种现场快速便携式检测装置。基于前期开发的新型管中管耗材,本团队建立了基于肉眼或智能手机识别检测结果的单样本便捷核酸检测方法,并开发设计了多样本同时自动化检测的便携式小型化装置以及基于同轴光纤的荧光检测光路。基于上述方法和装置对新型冠状病毒核酸进行检测,检测灵敏度低于10 copy/μL,检测时间可缩短至32 min,检测过程中无需开盖,适用于家庭自检或现场快速检测。

Objective Clustered regularly interspaced short palindromic repeats(CRISPR)has shown significant promise as an e merging nucleic acid detection technology.However,it still requires improvement in terms of sensitivity,detection automation,and anti-pollution.Furthermore,CRISPR technology lacks simple and portable professional equipment to meet the high demand of rapid point-of-care testing.Therefore,this study proposes a CRISPR/Cas12a detection reaction system for SARS-Co V2.-This detection response system and innovative tube-in-tube consumables aid in developing a portable compact device for simultaneous automatic detection of several samples and a coaxial fiber-based fluorescence detection system.Finally,we developed a single-sample user-friendly nucleic acid detection APP based on smartphone recognition and detection results for the manual detection mode.Methods The target in this study was severe acute respiratory syndrome coronavirus-2(SARS-Co V2-),which was d etected using the CRISPR method and enhanced via the reverse transcription-recombinase polymerase amplification(RTRPA)technique;the feasibility was assessed using the reverse transcription-polymerase chain reaction(RT-PCR)amplification method in the early stages.Various companies customized the required reagents and the designed sequences.In the detection process,first,with the tube-in-tube consumables developed by our team in the early stage,which comprised the reaction outer and inner tubes,the amplification reagents and detection reagents were loaded into the inner and outer tubes,respectively.The temperature was regulated to 37--42℃to complete the amplification.The reagents in t he inner and outer tubes were then mixed by shaking or centrifugation,and the temperature was adjusted to complete the CRISPR reaction.Finally,it was possible to observe if there was any fluorescence occurrence under the illumination of a blue light.The detection instrument was composed of an optical cassette and a base,and automatic detection was realized through a printed circuit board(PCB),a human-computer interaction display screen,etc.In addition,this study also used the fluorescence image recognition algorithm to process the detection images,compared with the international standard polymerase chain reaction(PCR)technology to explore the detection limit,and increased the target types to test the specificity strength.Results and Discussions The lower part of the detection instrument designed by our team integrates the printed circuit b oard and the human-computer interaction display screen.In the automatic detection mode,the fluorescence recognition circuit was designed with the help of a 470 nm light-emitting diode(LED),an optical filter,a complementary metal oxide semiconductor(CMOS)camera,a collimating lens,and a coaxial fiber.At the same time,the specificity of the theoretical experiment was verified through comparative experiments on several different targets.In addition,to verify the accuracy of this method for detecting actual samples,we compared each actual sample through PCR detection and the method based on the combination of RT-RPA and CRISPR proposed in this study.The detection results showed that the two were perfectly consistent.Conclusions The current study proposed a CRISPR/Cas12a-based anti-pollution portable nucleic acid detection t echnique.Furthermore,a simple model was proposed based on the naked eye or smartphone to recognize results;additionally,a downsized portable device based on fluorescence detection that can simultaneously detect numerous samples was constructed.The portable device can detect numerous samples simultaneously,and it has a constant heating mechanism and fluorescence stimulation detection optical channel to enhance the detection system’s accuracy and stability.The nucleic acid of SARS-Co V2-was verified using the proposed method and detection system.The minimum detection limit was<10 copy/μL.The test findings of our method had a good consistency with that of real-time f luorescence quantitative PCR method,but our method took less than half the time consuming of the PCR method,and the whole detection process could be finished in 32 min.The method and technology developed in this study propose a novel approach for nucleic acid detection at health-care center and home.