Development of a visual detection method of porcine deltacoronavirus using loop-mediated isothermal amplification

Abstract

The emergence of porcine deltacoronavirus (PDCoV) presents a significant threat to both human and animal health due to its ability to cause highly contagious enteric diseases. This underscores the crucial need for timely and accurate diagnosis to facilitate effective epidemiological investigation and clinical management. This research aimed to establish a visual detection method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for PDCoV testing. In this study, six pairs of primers were designed according to the conserved sequences of PDCoV ORF1a/b genes. The primer sets and parameters that affect LAMP reaction were optimized. The visual RT-LAMP method was developed by incorporating methyl red into the optimized reaction system, it exclusively detected PDCoV without cross-reactivity with other viruses and the detection limits for PDCoV could reach 10 copies/μL. In comparison with RT-PCR for testing 132 clinical samples, the relative specificity and sensitivity of the visual RT-LAMP were found to be 99.2 and 100%, respectively, with a concordance rate of 99.2% and a kappa value of 0.959, indicating that the visual RT-LAMP is a reliable method for the application of PDCoV detection in clinical samples.

Keywords: deltacoronavirus; detection; loop-mediated isothermal amplification; porcine; visualization.

Figure 1

Screening results of PDCoV RT-LAMP primer sets. M for 2,000 bp DNA marker, lanes 1 and 2 represent positive and negative amplification results of primer sets 1, respectively; lanes 3 and 4 for primer sets 2; lanes 5 and 6 for primer sets 3; lanes 7 and 8 for primer sets 4; lanes 9 and 10 for primer sets 5; lanes 11 and 12 for primer sets 6.

Figure 2

Optimization of the PDCoV RT-LAMP reaction system. (A) Reaction time; (B) reaction temperature; (C) inner primer concentration; (D) outer primer concentration; (E) Bst enzyme concentration; (F) Mg²⁺ concentration; (G) dNTPs concentration; (H) Buffer concentration. The final optimized conditions are as follows: reaction 50 min (A) at 60°C (B), the concentrations of inner primer and outer primer were 48 μmol/L (C) and 5 μmol/L (D), respectively. The optimal concentrations of Bst enzyme, Mg²⁺, dNTPs and buffer were found to be 4,000 U/mL (E), 100 mmol/L (F), 10 mmol/L (G), and 1.4× (H), respectively.

Figure 3

Establishment of visual PDCoV RT-LAMP assay. (A) The results of RT-LAMP gel electrophoresis and visual RT-LAMP with methyl red indicator; (B) specificity evaluation of the visual RT-LAMP assay.

Figure 4

Sensitivity evaluation of the visual RT-LAMP assay. (A) The results of RT-LAMP gel electrophoresis; (B) the results of RT-LAMP with methyl red indicator; (C) the results of RT-PCR gel electrophoresis: M for 2,000 bp DNA marker, lanes 1–6 represent DNA marker, lanes 1–9 represent 10⁸–1 copies/μL standard plasmid; lane 10 represents negative control.