基于RAA-CRISPR/EsCas13d的猪繁殖与呼吸综合征病毒II型的可视化快速检测方法

doi:10.13560/j.cnki.biotech.bull.1985.2025-1060

摘要/Abstract

摘要：

目的针对猪繁殖与呼吸综合征病毒Ⅱ型（PRRSV-2）现有检测方法在应用中存在操作复杂、检测周期长等问题，构建一种操作简便、反应快速的核酸检测方法，以满足疾病监测与疫情防控的需求。方法基于PRRSV-2基因组的保守区域设计引物和CRISPR RNA（crRNA），建立重组酶聚合酶等温扩增（RAA）与CRISPR/EsCas13d相结合的一锅式检测体系。通过对反应组分浓度及反应条件进行系统优化，实现RAA扩增、T7转录及CRISPR/EsCas13d切割反应在同一反应管内连续完成，避免反应过程中的开盖操作。检测结果可通过荧光仪器读取，亦可通过管内荧光信号进行肉眼可视化判读，并对该方法的灵敏度、特异性及临床样本检测性能进行评价，同时与RT-qPCR方法进行对比分析。结果建立的一锅式RAA-CRISPR/EsCas13d检测平台可在60 min内完成PRRSV-2的核酸检测。灵敏度分析结果显示，采用荧光仪器读取时，该方法对PRRSV-2 RNA的检测下限为8 copies/μL；采用肉眼可视化判读时，检测下限为50 copies/μL。特异性分析表明，该方法与经典猪瘟病毒、猪流行性腹泻病毒等常见猪源病毒无交叉反应。在56份临床样本检测中，该方法的检测结果与RT-qPCR方法完全一致。结论建立的一锅式RAA-CRISPR/EsCas13d检测平台可在60 min内完成PRRSV-2的灵敏核酸检测，并支持荧光仪器读取与肉眼可视化2种结果判读方式，为PRRSV-2的快速检测和疾病监测提供了操作简便的分子检测方案。

关键词: 猪繁殖与呼吸综合征病毒, 美洲型PRRSV, CRISPR/EsCas13d, 重组酶聚合酶扩增, 一锅式检测, 可视化检测

Abstract:

Objective To address the drawbacks in field applications, such as complex operation and long detection cycles, for porcine reproductive and respiratory syndrome virus type Ⅱ (PRRSV-2), this study aimed to develop a rapid and convenient detection method for disease surveillance and epidemic control. Method Primers and CRISPR RNAs (crRNAs) were designed based on conserved regions of the PRRSV-2 genome. A one-pot detection system integrating recombinase-aided amplification (RAA) and CRISPR/EsCas13d was established. Through systematic optimization of reaction component concentration and reaction conditions, RAA amplification, T7 transcription, and CRISPR/EsCas13d cleavage were sequentially completed in a single reaction tube, eliminating tube-opening steps during the assay. Detection results were obtained using a fluorescence instrument or by direct naked-eye visualization of in-tube fluorescence signals. Sensitivity, specificity, and clinical performance were evaluated and compared with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Result The one-pot RAA-CRISPR/EsCas13d detection platform enabled nucleic acid detection of PRRSV-2 within 60 min. Sensitivity analysis showed a detection limit of 8 copies/μL with fluorescence readout and 50 copies/μL with naked-eye visualization. Specificity analysis demonstrated no cross-reactivity with common porcine viruses, including classical swine fever virus and porcine epidemic diarrhea virus. For 56 clinical samples, detection results were fully consistent with those obtained by RT-qPCR. Conclusion The one-pot RAA-CRISPR/EsCas13d detection platform enables sensitive detection of PRRSV-2 within 60 min. This platform supports fluorescence-based readout and naked-eye visualization, and provides a simple and rapid molecular diagnostic method for PRRSV-2 detection and disease surveillance.

Key words: porcine reproductive and respiratory syndrome virus, PRRSV-2, CRISPR/EsCas13d, recombinase-aided amplification, one-pot detection, visual detection

赵磊, 戴阳明, 何姝颖, 朱玲, 徐志文. 基于RAA-CRISPR/EsCas13d的猪繁殖与呼吸综合征病毒II型的可视化快速检测方法[J]. 生物技术通报, 2026, 42(8): 81-89.

ZHAO Lei, DAI Yang-ming, HE Shu-ying, ZHU Ling, XU Zhi-wen. A Rapid and Visual Detection Method for Porcine Reproductive and Respiratory Syndrome Virus Type II （PRRSV-2） Based on the RAA-CRISPR/EsCas13d[J]. Biotechnology Bulletin, 2026, 42(8): 81-89.

图/表 4

图1 RAA-EsCas13d病毒检测平台的设计流程及原理

Fig. 1 Design workflow and principle of the RAA-EsCas13d virus detection platform

图2 EsCas13d 蛋白的SDS-PAGE和Western blot结果A：SDS-PAGE结果；B：Western blot结果；M：蛋白质分子质量标准；1：阴性对照；2：EsCas13d蛋白

Fig. 2 SDS-PAGE and Western blot results for the EsCas13d proteinA: SDS-PAGE results. B: Western blot results. M: Protein molecular weight marker. 1: Negative control. 2: EsCas13d protein

图4 RAA引物与crRNA的筛选A：9对RAA引物的琼脂糖凝胶电泳结果；B：9对RAA引物的单管RAA-EsCas13d复核结果；C：3个crRNA的荧光强度对比（n=3；误差线代表平均值±SD；ns：无统计学意义；*P<0.05）；D：3个crRNA的荧光曲线

Fig. 4 Screening of RAA primers and crRNAsA: Agarose gel electrophoresis of 9 RAA primer pairs. B: Single-tube RAA-EsCas13d verification of 9 primer pairs. C: Comparison of fluorescence intensities of 3 crRNAs (n=3; ns: no statistical significance; error bars represent mean value ± SD; * P<0.05). D: Fluorescence curves of 3 crRNAs

图5 单管RAA-CRISPR/EsCas13d的建立及其优化A，B：两步法及优化前后单管RAA-CRISPR/EsCas13d的灵敏度分析；C：crRNA和EsCas13d蛋白浓度优化；D：KCl浓度及pH值优化；E：单管RAA-CRISPR/EsCas13d的特异性评估；F、G：单管RAA-CRISPR/EsCas13d特异性和灵敏度的可视化检测（n=3；误差线代表平均值±SD；*P<0.05，**P<0.01，***P<0.001，****P<0.000 1）

Fig. 5 Establishment and optimization of single-tube RAA-CRISPR/EsCas13dA, B: Sensitivity comparison between the two-step method and the single-tube RAA-CRISPR/EsCas13d assay before and after optimization. C: Optimization of crRNA and EsCas13d protein concentrations. D: Optimization of KCl concentration and pH conditions. E: Specificity evaluation of the single-tube RAA-CRISPR/EsCas13d assay. F, G: Visual readout of the specificity and sensitivity of the single-tube RAA-CRISPR/EsCas13d assay (n=3; error bars represent mean value ± SD; *P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1)

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