Development and application of a CRISPR/Cas12a-based reverse transcription–recombinase polymerase amplification assay with lateral flow dipstick and fluorescence detection for Getah virus

Viruses and clinical samples

Getah virus (GETV), Japanese encephalitis virus (JEV), pseudorabies virus (PRV), porcine circovirus (PCV), and classical swine fever virus (CSFV) sera were provided by the Military Veterinary Research Institute, Academy of Military Medical Sciences (Changchun, China). For clinical validation, 30 porcine serum samples were collected from a swine farm affiliated with the Jinzhou Medical University in Liaoning Province (Experimental Animal Ethics Committee of Jinzhou Medical University Approval no. 2024176-5.

Design of primers, crRNAs, and ssDNA reporters

The E2 protein gene sequences of GETV (NCBI Gene IDs: EU015063, EF631998, EU015061, EU015062, KY434327, KY450683, and MG869691) were aligned using MEGA 6.06, selecting the conserved region of GETV (EU015063) E2 protein from 179 bp–479 bp as the target gene to design two crRNAs, each paired with two primers. Two types of ssDNA reporters were synthesized: a fluorescence reporter (5′-FAM-TTATT-BHQ1-3′) for CRISPR-Cas12a-based fluorescence detection and an LFD reporter (5′-FAM-TTTTTTATTTTTT-Biotin-3′) for LFD-based visualization. The reverse transcription qualitative polymerase chain reaction (RT-qPCR) primers used to simulate the clinical assay were based on the GETV RT-qPCR assay established by Cao et al. (2022). All primers, crRNAs, and ssDNA reporters (Table 1) were synthesized by Sangon Biotech (Shanghai, China).

Construction of standard plasmid

First, extract RNA from the serum containing GETV using the TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China), and reverse transcribe the RNA into cDNA to use the cDNA of GETV as a template for PCR amplification. The polymerase chain reaction (PCR) system was 50 µL (50 µL reaction mixture: 25 µL 2 × Taq Mix (Seven Biotech), two µL GETV-PCR upstream and downstream primers (10 µM) (Table 1), two µL template, 21 µL ddH2O) under the following conditions: 94 °C for 2 min; 35 cycles of 94 °C (45 s), 56 °C (45 s), 72 °C (45 s) and final extension at 72 °C for 10 min. The PCR products were verified via 1.2% agarose gel electrophoresis, purified using the SanPrep DNA Gel Extraction Kit (Sangon Biotech), and cloned into the pEASY-T1 vector (TransGen Biotech, Beijing, China). Recombinant plasmids were transformed into DH5α-competent cells, single white colonies were screened after overnight incubation using solid medium containing kanamycin sulphate, and the white colonies were selected and plasmids were extracted using the Plasmid Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). The extracted standard plasmids were sequenced and verified by Sangon Biotech. The plasmid concentration was measured by a micro UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the copy number per unit volume of plasmid was calculated by formula and stored at −20 °C:

Plasmid copy number (copies/µL)=[plasmid concentation (g/µL)×10-9 ×6.02 ×1023]/

    {[Vector length (bp) + fragment length (bp)]×660/gmol}.

Establishment of RT-RPA assay

RT-RPA reactions were performed using the commercially available RT-RPA Nucleic Acid Amplification Kit (Gendx Biotech, Suzhou, China) according to the manufacturer’s specifications. In brief, 50 µL of reaction mixture contained 20 µL of rehydration buffer, 2.5 µL of each primer (10 µM), three µL of template, 20 µL of ddH2O, and two µL of the activator(magnesium acetate). A 48 µl premix of the reactants except activator was first prepared, and then the premix was transferred to a tube containing lyophilised enzyme RT-basic amplification reagent. After shaking and mixing, two µl of activator was added to the cap of the reaction tube, and the activator was briefly centrifuged into the premix. Finally, the reaction tube was incubated in a thermostat at 42 °C for 20 min. The amplified product (20 µL) was mixed with 40 µL of phenol, centrifuged, and screened for the best RT-RPA primers by 1.2% agarose gel electrophoresis.

Fluorescence-based RT-RPA-CRISPR/Cas12a detection (FBDA)

The CRISPR-Cas12a fluorescence assay (total 20 µL) included one µL of crRNA (10 µM), two µL of 10 × HOLMES buffer (TOLOBIO, Shanghai, China), 0.5 µL of LbCas12a nuclease (10 µM; TOLOBIO, Shanghai, China), one µL of ssDNA reporter (10 µM), 13 µL of nuclease-free water, and 2.5 µL of RPA product. The reaction mixture was incubated with QuantStudio1 (ABI, Natick, MA, United States) at 37.5 °C for 20 min, and the fluorescence (FAM) signal was detected every 20 s. The reaction tubes can be placed under an LED UV transmission unit (Tanon, Shanghai, China) to observe the fluorescence intensity with the naked eye after the test. Screening crRNA by FBDA

RT-RPA-CRISPR/Cas12a-LFD assay

For lateral flow detection, the CRISPR-Cas12a reaction mixture was diluted to 50-µL volume with nuclease-free water and applied to an LFD strip (Gendx Biotech, Suzhou, China) (results appear in 3 min and are valid within 10 min). The results were interpreted as follows: dual red bands (control line (C) and test line (T)) indicated positivity; a single C band indicated negativity. Strips lacking a C band were deemed invalid and retested.

Sensitivity and specificity testing

The constructed plasmids were diluted to 10⁶–10⁰copies/µL and then tested for sensitivity using FBDA and RT-RPA-CRISPR/Cas12a-LFD assays, respectively, and the fluorescence intensity could be observed visually by placing the reaction tubes under the LED UV projection after the FBDA assay. The specific detection method was consistent with the sensitivity detection method, but the samples selected for the specific detection were nucleic acids extracted from viral serum (GETV, JEV, PRV, PCV and CSFV), and the DNA of PRV and PCV, as well as the RNA of GETV, JEV and CSFV, were extracted using the TIANamp Viral DNA/RNA Kit (Tiangen, Beijing, China), and the RNA was reverse transcribed into cDNA. Nucleic acid concentrations were measured by a micro-UV-Vis spectrophotometer (Thermo Fisher Scientific) and the five nucleic acids were diluted to the same concentration of 50 ng/µL. All of the above assays were repeated three times to ensure the feasibility and reproducibility of the experiment.

Validation with simulated clinical samples

No cases of infection by GETV have been reported in Liaoning province, China. Due to the scarcity of true positive samples, this study used a simulated positive sample strategy to comparatively analyse the accuracy between RT-RPA-CRISPR/Cas12a-LFD and reverse transcription qualitative polymerase chain reaction (RT-qPCR). Six tubes of serum containing GETV were randomly and blindly mixed into 30 clinical samples to form a sample containing 36 cases. All samples were completed by independent operators with unknown grouping information. All samples were subjected to RT-qPCR according to the method of Cao et al. (2022) for comparison with the results of RT-RPA-CRISPR/Cas12a-LFD assay.

Successful construction of an RT-RPA-CRISPR/Cas12a system for GETV detection (Fig. 1)

The CRISPR-Cas12a detection architecture integrates recombinant LbCas12a nuclease, CRISPR array-optimized crRNA guides, fluorogenic ssDNA reporters, and RPA-amplified dsDNA targets. Leveraging the programmability of CRISPR/Cas12a, crRNA was designed to base-pair with the conserved E2 gene of GETV. The integration of RT-RPA with CRISPR/Cas12a enhanced the detection sensitivity. In this system, preincubated Cas12a–crRNA complexes specifically recognize and cleave target dsDNA amplified via RT-RPA, activating the trans-cleavage activity of Cas12a to indiscriminately degrade ssDNA probes. Fluorescently quenched ssDNA probes served as reporters, and target-induced cleavage restored the fluorescence signals for quantitative detection. Additionally, an LFD assay was developed using FAM- and biotin-modified single-stranded DNA (ssDNA) reporters. In the negative samples, anti-FAM antibodies immobilized on the control (C) line captured uncleaved FAM-biotin reporters bound to gold nanoparticle-conjugated streptavidin. For the positive samples, the cleavage of reporters reduced the C-line intensity while enabling the accumulation of gold–streptavidin complexes at the test (T) line. The entire LFD readout was completed within 10 min, allowing for visual interpretation in field applications.

Screening of the RT-RPA primers and crRNA

Optimal RT-RPA primers and crRNA were selected through agarose gel electrophoresis and RT-RPA–CRISPR/Cas12a fluorescence assays. According to the brightness of the target band and the amplification efficiency of the fluorescence amplification curve in the agarose gel electrophoresis image, the brightest band of interest in the agarose gel electrophoresis image (Fig. 2A) was selected as the best RT-RPA primer (GETV-RPA-F1 and GETV-RPA-R A1), and the group with the highest amplification efficiency of the fluorescence amplification curve (Fig. 2B) was selected as the best crRNA (crRNA A), that is, crRNA A was selected and the first pair of primers (GETV-RPA-F1 and GETV-RPA-R A1).

Sensitivity of the RT-RPA-CRISPR/Cas12a system

The sensitivity of the RT-RPA-CRISPR/Cas12a system was evaluated using serially diluted GETV standard plasmids (10⁶–10⁰ copies/µL) and nuclease-free water as a negative control. Following RPA amplification, CRISPR/Cas12a-mediated fluorescence detection achieved a limit of detection of 10¹ copies/µL across triplicate reactions (Fig. 3A). Parallel testing with the LFD confirmed visible T-line signals at 10¹ copies/µL (Fig. 3B). Moreover, the detection sensitivity was repeated three times by LFD method, which proved the reproducibility of the experiment (Fig. 1S), validating the system’s high sensitivity.

Specificity of the RT-RPA-CRISPR/Cas12a system

The specificity of the assay was validated against four common swine pathogens: JEV, PRV, PCV, and CSFV. Both the fluorescence-based and LFD assays showed no cross-reactivity (Figs. 4A and 4B) (Fig. 2S), confirming the method’s high specificity for GETV.

Detection of simulated clinical samples

To assess clinical utility, 36 simulated samples were analyzed using the RT-RPA-CRISPR/Cas12a-LFD assay and compared with RT-qPCR. Both methods identified six positive samples with 100% concordance (Fig. 5, Table 2). The RT-RPA-CRISPR/Cas12a-LFD assay completed detection within 50 min.