Establishment of RPA-Cas12a-Based Fluorescence Assay for Rapid Detection of Feline Parvovirus

The feline parvovirus (FPV) used in this study was isolated from the fecal specimen from a domestic cat with clinical signs of suspected parvovirus infection in 2021 in Shanghai and validated by the Shanghai Animal Disease Control Center (SHADCC) according to a previous study (Bergmann et al. 2021). The FPV was seeded with F81 cells, cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco™, Thermo Fisher Scientific, Inc., USA) supplemented with 10% fetal bovine serum (FBS) with incubation at 37°C in 5% CO₂, and then stored in SHADCC. In contrast, other viruses were purchased from the American Type Culture Collection (ATCC^®, USA). Sixty anal swabs samples were collected from felines with clinical signs suspicious of feline panleukopenia and treated at the animal hospital or healthy felines in Shanghai and kept at −80°C.

According to the manufacturer’s recommendations, total DNA was extracted from FPV-infected F81 cells using AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen^®, Corning, USA). In contrast, to prepare the total viral DNA of clinical samples, 500 μl of sterile phosphate-buffered saline (PBS) was added to each tube with the anal swabs sample, vortexed for 5 min, and then centrifuged at 12,000 rpm for 15 min at 4°C. Subsequently, the supernatants were transferred to the microcentrifuge tube and utilized for viral DNA extraction using the kit described above. All extracted DNA samples were suspended in 50 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6 ), and stored at −80°C until further use.

To obtain the FPV-specific conserved sequences, genome sequences of fifteen strains of FPV (GenBank numbers: MW926316.1, MW926315.1, MW926314.1, MT614366.1, MT614366.1, MZ362883.1, MZ913313.1, MZ913314.1, MZ913315.1, MZ913316.1, MZ913317.1, MZ913318.1, MZ913319.1, OK128324.1, OK128325.1) were downloaded and aligned using ClustalW. After multiple alignments, the 404 bp fragment of the nonstructural gene NS1 was identified as the target template for molecular detection and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (China) and then cloned into pBluscript II SK (+) vector (Fig. 1B) to construct the recombinant plasmid pBluscript-NS1. Subsequently, the resulting plasmid was transformed into Escherichia coli TOP10 cells, allowing us to prepare the NS1 detection template for FPV conveniently. The number of DNA copies was determined using the following equation: DNAcopynumber=[M(ng/μl)×6.02×1023](N×109×660) $$DNA\,copy\,number = {{[M(ng/\mu l) \times 6.02 \times {{10}^{23}}]} \over {(N \times {{10}^9} \times 660)}}$$ where M indicates the concentration of DNA in nanograms measured using B-500 Biophotometer (Metash Instruments, China), N represents the length of the DNA in base, and the average molecular weight of a base is defined as 660 Da.

Based on the 404 bp fragment of the NS1 gene identified as the target fragment for FPV detection according to the results described above, four pairs of primers and different primer pairs (NS1-F1/NS1-R1, NS1-F2/NS1-R2, NS1-F3/NS1-R3, NS1-F4/NS1-R4, NS1-F3/ NS1-R1, NS1-F2/ NS1-R4, NS1-F1/ NS1-R3) for RPA amplification were designed according to the principles of RPA primer design, and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (China). Meanwhile, the specificity of RPA primers was assessed using Nucleotide BLAST online (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Then, different primer pairs were examined using the kit (LeSun Bio, China) to screen for the optimal primer pair. The optimal primer pair was applied in subsequent experiments. Briefly, RPA reaction mixtures were composed of 25 μl of 2 × reaction buffer, 2 μl of each primer (10 μM), 5 μl of DNA template (1 × 10⁴ copies/μl), 13 μl of nuclease-free water and 3 μl of magnesium acetate (280 mM). Then RPA reaction was conducted at 37°C for 25 min in an Axxin T8-isothermal instrument (T8-ISO; Axxin Pty., Ltd., Australia). The amplified products were resolved on a 1% (w/v) agarose gel and visualized under UV light.

To ensure the high efficiency of RPA-Cas12a-fluorescence assay for FPV, five crRNA sequences were designed based on the 404 bp fragment of the NS1 gene, and their corresponding primer pair for crRNA in vitro synthesis was synthesized by Sangon Biotech (Shanghai) Co., Ltd. (China). For crRNA preparation, their corresponding primer pairs for crRNA preparation in vitro were synthesized by GENEWIZ Germany GmbH (Germany) and annealed to produce the double-stranded DNA (dsDNA) using an annealing buffer. The generated dsDNA was extracted using the TIANgel Midi Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd., China) and used as the in vitro transcription reaction template (IVT). IVT was carried out using the IVT T7 Kit (Takara Biomedical Technology (Beijing) Co., Ltd., China) at 37°C. The IVT reaction mixture consisted of 5 μl of 10 × transcription buffer, 5 μl of each NTP solution (10 μM), 1 μl of RNase inhibitor (1 μM), 5 μl of T7 RNA polymerase (1 μM), 6.5 μl of RNase-free water and 6.25 μl of each single-stranded primer (10 μM). After 2 hours of incubation, a final concentration of 10 units of DNase I was added to the reaction mixture and further incubated at 37°C for 30 minutes to digest the template dsDNA fully. Subsequently, the crRNA was collected by centrifugation at 15,000 rpm for 20 min at 4°C, and the crRNA concentration was measured using B-500 Biophotometer. To screen for the optimal crRNA, crRNA target sites in the 404 bp fragment of NS1 gene were presented in Fig. 1A. Details of primer sets and ssDNA reporter are shown in Table I.

The specificity and sensitivity examination of the RPA-Cas12a-fluorescence assay for FPV was conducted under the optimized reaction conditions described above. RPA-amplified products were briefly added to the CRISPR/ Cas12a reaction mixture. Then 5 μl of RPA products were added to 20 μl of the CRISPR/Cas12a reaction mixture, which consisted 2 μl of crRNA (80 nM), 0.5 μl of Cas12a (20 nM; New England Biolabs, UK), 4 μl of ssDNA reporter (160 nM), 2.5 μl of 10 × NEB 2.1 buffer (New England Biolabs, UK) and 11 μl of RNase-free water. Finally, the reaction mixtures were incubated in the Axxin T8-isothermal instrument for 20 min at 37°C to evaluate fluorescent signals.

For the sensitivity assessment, the detection limit was evaluated using the 10-fold dilutions of the recombinant plasmid pBluscript-NS1 ranging from 100 to 0.1 copies/μl as the reaction template for the RPA-Cas12a-fluorescence assay. The limit of detection was determined according to the reaction consisting of the minimum amount of DNA template that produced a fluorescent signal. For the specificity analysis, each DNA or cDNA sample of 1.0 × 10⁴ copies/μl from feline calicivirus/Cat/Shanghai/01/2021 (FCV), feline herpesvirus-1/Cat/Shanghai/01/2014 (FHV-1), feline infectious peritonitis virus ATCC^® VR-990™ (FIPV), feline Mycoplasma felis ATCC^® 23391™ (MYC), and Chlamydia psittaci ATCC^® VR-120™ (CP) were applied as the templates for the RPA-Cas12a-fluorescence assay. FCV and FHV-1 strains were previously isolated from nasal swabs of feline in the animal relief center in Shanghai and stored by SHADCC. FIPV, MYC, and CP were bought from the American Type Culture Collection (ATCC^®, USA).

The viral DNA was extracted from clinical samples and performed using RPA-Cas12a-fluorescence assay. The results were compared with those obtained by qPCR as previously described (Van Brussel et al. 2019). The assay was carried out using the ABI 7500 instrument (Applied Biosystems).

The level of agreement between the two methods was determined using Cohen’s “kappa” (κ) analysis via the DiagnosticTest using openepi software (Dean et al. 2013), showcasing a robust agreement with a κ-value of ≥ 0.750, coupled with a highly significant p-value of < 0.0005.