基于CRISPR/Cas12a技术的猪流行性腹泻病毒检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2026-0272

摘要/Abstract

摘要：

目的建立逆转录环介导等温扩增（RT-LAMP）与CRISPR/Cas12a系统联合检测体系，为猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）的快速诊断提供新的技术手段。方法基于PEDV M基因保守序列区域设计特异性crRNA、PCR及LAMP引物，通过筛选确定最优crRNA，建立并优化LAMP-CRISPR/Cas12a检测体系，利用JOE和ROX荧光报告分子实现可视化检测。采用实时荧光定量PCR（RT-qPCR）、PCR-CRISPR/Cas12a和LAMP-CRISPR/Cas12a三种检测方法进行灵敏度对比分析，评估检测系统的特异性。最后利用临床粪便样本验证所建立检测方法的准确性。结果建立的RT-LAMP-CRISPR/Cas12a检测系统集前处理、核酸扩增、Cas12a介导的特异性识别与荧光信号转换于一体，可在80 min内实现PEDV的可视化检测，适用于粪便样本的全流程检测。针对目标质粒的检测灵敏度为9.03×10^-7 copies/μL，相比RT-qPCR和PCR-CRISPR/Cas12a检测方法的灵敏度提高了10个数量级。特异性评估结果表明仅目标病毒产生强荧光信号。采用RT-LAMP-CRISPR/Cas12a和RT-PCR-CRISPR/Cas12a系统检测30份猪粪便样本，两种方法均检出9个阳性样本，检出率为30%，结果一致性为100%。结论成功构建了RT-LAMP-CRISPR/Cas12a检测PEDV的技术体系，该体系具有高灵敏度、强特异性、快速高效、操作简便等优势，能够满足快速检测的应用需求。

关键词: 猪流行性腹泻病毒, RT-LAMP, CRISPR/Cas12a, 核酸检测, 可视化

Abstract:

Objective The development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with the CRISPR/Cas12a system provides a new technological approach for the rapid diagnosis of porcine epidemic diarrhea virus (PEDV). Method Specific crRNAs, PCR, and LAMP primers targeting the conserved region of the PEDV M gene were designed. The optimal crRNA was screened and determined, and the LAMP-CRISPR/Cas12a detection system was established and optimized. Visual detection was enabled using JOE and ROX fluorescent reporters. Sensitivity comparison analysis was conducted among real-time fluorescence quantitative PCR (RT-qPCR), PCR-CRISPR/Cas12a, and LAMP-CRISPR/Cas12a detection methods, while the specificity of the detection systems was assessed. The clinical applicability of the established detection method was validated using clinical fecal samples. Result An RT-LAMP-CRISPR/Cas12a detection system has been developed that combines sample pre-processing, nucleic acid amplification, and Cas12a-mediated specific identification, as well as fluorescence signal conversion. This system allows for visual detection of PEDV within 80 minutes and is suitable for complete testing of fecal samples. The detection sensitivity for the target plasmid is 9.03×10^-7 copies/μL, representing a 10-fold increase in sensitivity compared to RT-qPCR and PCR-CRISPR/Cas12a detection methods. Specificity assessment results indicated strong fluorescence signals only from the target virus. Application of the RT-LAMP-CRISPR/Cas12a and RT-PCR-CRISPR/Cas12a systems on 30 porcine fecal samples revealed nine positive samples using both methods, yielding a detection rate of 30% with 100% consistency in results. Conclusion The RT-LAMP-CRISPR/Cas12a detection system for PEDV has been successfully established. This system offers high sensitivity, strong specificity, rapid efficiency, and ease of operation, making it suitable for rapid testing applications.

Key words: porcine epidemic diarrhea virus, RT-LAMP, CRISPR/Cas12a, nucleic acid detection, visualization

李聪聪, 孙长胜, 徐秋良. 基于CRISPR/Cas12a技术的猪流行性腹泻病毒检测方法的建立[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0272.

LI Cong-cong, SUN Chang-sheng, XU Qiu-liang. Establishment of a Detection Method for Porcine Epidemic Diarrhea Virus Based on CRISPR/Cas12a Technology[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0272.

图/表 8

图1 最适crRNA的筛选A：不同激发光下 crRNA的LbCas12a酶切图；B： LbCas12a酶切检测终点收集的荧光信号值。NC为阴性对照。**P<0.01，下同

Fig. 1 Optimal crRNA selectionA: Cleavage patterns of crRNA by LbCas12a enzyme under different excitation lights. B: The fluorescence signal values were collected at the endpoint of the LbCas12a enzyme cleavage assay. NC represents the negative control. **P<0.01, the same below

图2 反应条件优化A：LAMP最适反应温度筛选；B：LAMP最适反应时间筛选；C： ROX探针最适LbCas12a酶酶切时间筛选；D：JOE探针最适LbCas12a酶酶切时间筛选。NC为阴性对照，M为DL2000 marker

Fig. 2 Optimization of reaction conditionsA: Screening for the optimal reaction temperature of LAMP. B: Screening for the optimal reaction time of LAMP. C: Optimization of the cleavage time of the LbCas12a enzyme with the ROX probe. D: Optimization of the cleavage time of the LbCas12a enzyme with the JOE probe. NC represents the negative control, while M denotes DL2000 marker

图3 猪流行性腹泻病毒荧光定量PCR灵敏度检测A：标准曲线；B：扩增曲线；C：梯度稀释比例质粒的Ct值。1-7对应的质粒模板浓度分别为9.03×109、9.03×108、9.03×107、9.03×106、9.03×105、9.03×104、9.03×103 copies/μL

Fig. 3 Sensitivity assessment of porcine epidemic diarrhea virus detection through real-time fluorescent quantitative PCRA: Standard curve. B: Amplification curve. C: The Ct values of a plasmid dilution series. The plasmid template concentrations corresponding to 1-7 are 9.03×109, 9.03×108, 9.03×107, 9.03×106, 9.03×105, 9.03×104, and 9.03×103 copies/μL

图5 LAMP 结合CRISPR/Cas12a 系统的灵敏度检测A：LAMP-CRISPR/Cas12a-JOE各激发光下LbCas12a酶切结果；B：LAMP-CRISPR/Cas12a-ROX各激发光下LbCas12a酶切结果；C：PCR-CRISPR/Cas12a-JOE各质粒浓度荧光强度值；D：LAMP-CRISPR/Cas12a-ROX各质粒浓度荧光强度值。1-8扩增模板浓度依次为9.03×10-1、9.03×10-2、9.03×10-3、9.03×10-4、9.03×10-5、9.03×10-6、9.03×10-7、9.03×10-8 copies/μL

Fig. 5 Detection sensitivity of the LAMP combined with the CRISPR/Cas12a systemA: Cleavage results of LbCas12a enzyme under different excitation wavelengths for LAMP-CRISPR/Cas12a-JOE. B: Cleavage results of LbCas12a enzyme under different excitation wavelengths for LAMP-CRISPR/Cas12a-ROX. C: Fluorescence intensity values of various plasmid concentrations for PCR-CRISPR/Cas12a-JOE. D: Fluorescence intensity values of various plasmid concentrations for LAMP-CRISPR/Cas12a-ROX. Template concentrations 1-8 are 9.03×10-1, 9.03×10-2, 9.03×10-3, 9.03×10-4, 9.03×10-5, 9.03×10-6, 9.03×10-7, 9.03×10-8 copies/μL

图6 RT-LAMP/ LAMP 结合CRISPR/Cas12a系统的特异性检测A：RT-LAMP/LAMP-CRISPR/Cas12a-JOE各激发光下LbCas12a特异性酶切结果；B：RT-LAMP/LAMP-CRISPR/Cas12a-ROX各激发光下LbCas12a特异性酶切结果；C：RT-LAMP/LAMP-CRISPR/Cas12a-JOE各病毒荧光强度值；D：RT-LAMP/LAMP-CRISPR/Cas12a-ROX各种病毒荧光强度值。PEDV：猪流行性腹泻病毒，PRRSV：猪繁殖与呼吸综合征病毒，PRV：猪伪狂犬病毒，PPV：猪细小病毒

Fig. 6 Specific detection through the combination of RT-LAMP/LAMP and the CRISPR/Cas12a systemA: Specific cleavage results of LbCas12a under different excitation lights in RT-LAMP/LAMP-CRISPR/Cas12a-JOE. B: Specific cleavage results of LbCas12a under different excitation lights in RT-LAMP/LAMP-CRISPR/Cas12a-ROX. C: Fluorescence intensity values of various viruses in RT-LAMP/LAMP-CRISPR/Cas12a-JOE. D: Fluorescence intensity values of different viruses in RT-LAMP/LAMP-CRISPR/Cas12a-ROX. PEDV: Porcine epidemic diarrhea virus, PRRSV: Porcine reproductive and respiratory syndrome virus, PRV: Pseudorabies virus, PPV: Porcine parvovirus

图4 PCR结合CRISPR/Cas12a 系统的灵敏度检测A：PCR-CRISPR/Cas12a-JOE各激发光下LbCas12a酶切结果；B：PCR-CRISPR/Cas12a-ROX各激发光下LbCas12a酶切结果；C：PCR-CRISPR/Cas12a-JOE各质粒浓度荧光强度值；D：PCR-CRISPR/Cas12a-ROX各质粒浓度荧光强度值。1-10扩增模板浓度依次为9.03×109、9.03×108、9.03×107、9.03×106、9.03×105、9.03×104、9.03×103、9.03×102、9.03×101、9.03×100 copies/μL

Fig. 4 Detection of sensitivity in PCR combined with the CRISPR/Cas12a systemA: Enzymatic cleavage results of LbCas12a under different excitation wavelengths for PCR-CRISPR/Cas12a-JOE. B: Enzymatic cleavage results of LbCas12a under different excitation wavelengths for PCR-CRISPR/Cas12a-ROX. C: Fluorescence intensity values of various plasmid concentrations for PCR-CRISPR/Cas12a-JOE. D: Fluorescence intensity values of various plasmid concentrations for PCR-CRISPR/Cas12a-ROX. Template concentrations 1-10 are 9.03×109, 9.03×108, 9.03×107, 9.03×106, 9.03×105, 9.03×104, 9.03×103, 9.03×102, 9.03×101, 9.03×100 copies/μL

图7 猪流行性腹泻病毒RT-LAMP-CRISPR/Cas12a系统粪便样本检测A：粪便样本RT-LAMP-CRISPR/Cas12a-JOE不同激发光下LbCas12a酶酶切结果；B：粪便样本RT-LAMP-CRISPR/Cas12a-ROX不同激发光下LbCas12a酶酶切结果；C：粪便样本RT-LAMP-CRISPR/Cas12a-JOE荧光强度值；D：粪便样本RT-LAMP-CRISPR/Cas12a-ROX荧光强度值。1-30为粪便样本号，PC为阳性对照，NC为阴性对照

Fig. 7 Fecal sample detection of porcine epidemic diarrhea virus through RT-LAMP-CRISPR/Cas12a systemA: Results of LbCas12a enzyme cleavage under different excitation lights in fecal samples using RT-LAMP-CRISPR/Cas12a-JOE. B: Results of LbCas12a enzyme cleavage under different excitation lights in fecal samples using RT-LAMP-CRISPR/Cas12a-ROX. C: Fluorescence intensity values in fecal samples using RT-LAMP-CRISPR/Cas12a-JOE. D: Fluorescence intensity values in fecal samples using RT-LAMP-CRISPR/Cas12a-ROX. Sample numbers 1-30 represent fecal sample IDs. PC denotes positive control, and NC denotes negative control

图8 猪流行性腹泻病毒RT-PCR-CRISPR/Cas12a系统粪便样本检测A：粪便样本RT-PCR-CRISPR/Cas12a-JOE不同激发光下LbCas12a酶酶切结果；B：粪便样本RT-PCR-CRISPR/Cas12a-ROX不同激发光下LbCas12a酶酶切结果；C：粪便样本RT-PCR-CRISPR/Cas12a-JOE荧光强度值； D：粪便样本RT-PCR-CRISPR/Cas12a-ROX荧光强度值。1-30为粪便样本号，PC为阳性对照，NC为阴性对照

Fig. 8 Fecal sample detection of porcine epidemic diarrhea virus through the RT-PCR-CRISPR/Cas12a systemA: Results of LbCas12a enzyme cleavage under different excitation lights in fecal samples using RT-PCR-CRISPR/Cas12a-JOE. B: Results of LbCas12a enzyme cleavage under different excitation lights in fecal samples using RT-PCR-CRISPR/Cas12a-ROX. C: Fluorescence intensity values in fecal samples using RT-PCR-CRISPR/Cas12a-JOE. D: Fluorescence intensity values in fecal samples using RT-PCR-CRISPR/Cas12a-ROX. Sample numbers 1-30 represent fecal sample IDs. PC denotes positive control, and NC denotes negative control

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