A nucleic acid detection assay combining reverse transcription recombinase-aided amplification with a lateral flow dipstick for the rapid visual detection of porcine deltacoronavirus

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 °C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p < 0.001). Overall, the RT-RAA-LFD assay is a user-friendly diagnostic tool that can rapidly and visually detect PDCoV.

KEYWORDS:

• Detection
• lateral flow dipstick
• nucleic acid
• porcine deltacoronavirus
• recombinase-aided amplification

Author contributions

JZ performed the experiments. WW, LZ, XG, JH, XG, YC, and YZ analysed the experimental data. JZ and YZ wrote the paper. YZ designed the experiments. HY supervised experiments. All authors have read the manuscript and approved the submission.

Disclosure statement

No potential conflict of interest was reported by the author(s).

supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2022.2116157

Data availability statement

All relevant experimental data are available either in the text or in the supplementary materials. Detailed information for PDCoV, PEDV, PDCoV, TGEV, PRoV, PEAV, FMDV, PRRSV, PRV, and PCV2 are available to the public under GenBank accession numbers cited in Materials and Methods section.

Additional information

Funding

This study was funded by grants from the National Key R&D Program of China (No. 2021YFD1801103) and the earmarked fund for China Agriculture Research System (No. CARS-35).