Point-of-care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

Abstract

Porcine deltacoronavirus (PDCoV) has emerged and spread throughout the porcine industry in many countries over the last 6 years. PDCoV caused watery diarrhoea, vomiting and dehydration in newborn piglets. A sensitive diagnostic method would be beneficial to the prevention and control of PDCoV infection. Recombinase polymerase amplification (RPA) is an isothermal amplification method which has been widely used for virus detection. A probe-based reverse transcription RPA (RT-RPA) assay was developed for real-time detection of PDCoV. The amplification can be finished in 20 min and fluorescence monitoring was performed by a portable device. The lowest detection limit of the PDCoV RT-RPA assay was 100 copies of RNA molecules per reaction; moreover, the RT-RPA assay had no cross-reaction with other common swine viruses. The clinical performance of the RT-RPA assay was evaluated using 108 clinical samples (54 intestine specimens and 54 faecal swab specimens). The coincidence rate of the detection results for clinical samples between RT-RPA and RT-qPCR was 97.2%. In summary, the real-time RT-RPA assay offers a promising alternative to RT-qPCR for point-of-care detection of PDCoV.

Keywords: detection; porcine deltacoronavirus; recombinase polymerase amplification.

Figure 1

Primer sets screening. The products of RT‐RPA by the basic RPA kit using five primer sets were subjected to electrophoresis on a 2% agarose gel respectively

Figure 2

Alignment of the sequences of the primer‐probe set used in the real‐time RT‐RPA assay. The forward primer (F2) and probe were used as shown while the reverse primer (R2) was used as antisense oligonucleotides. Nucleotide sequences of the primers (F2/R2) and probe are shown at the top of the frames while the corresponding nucleotide sequences of other 25 PDCoV strains are shown at the bottom. Dots represent that nucleotides are identical to that of PDCoV strain CHN‐GD16‐02

Figure 3

Sensitivity and specificity of the real‐time RT‐RPA assay. (a) The sensitivity of the real‐time RT‐RPA assay. (b) The specificity of the real‐time RT‐RPA assay. SADS‐CoV, TGEV, PEDV, PRRSV, PRV, CSFV, PCV2, RV, PPV, FMDV, SIV and distilled water were the negative samples [Colour figure can be viewed at http://www.wileyonlinelibrary.com/]

Figure 4

Performance of the RT‐RPA assay. (a) Semi‐logarithmic regression of the data collected from eight runs using the RNA standard analyzed by GraphPad Prism 5.0. (b) Linear regression analysis of RT‐RPA threshold time (TT, y axis) and RT‐PCR cycle threshold (CT) values (x axis) were determined by Prism software. R ² value was 0.11