A Rapid and Visual Detection Method for Porcine Reproductive and Respiratory Syndrome Virus Type II （PRRSV-2） Based on the RAA-CRISPR/EsCas13d

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

Abstract

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

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.

Figures/Tables 4

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

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

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

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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