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

Abstract

Summary 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.

Rapid and visual detection of porcine deltacoronavirus by recombinase polymerase amplification combined with a lateral flow dipstick

“…In addition, the detection limit of the LFD-RPA assay was 10 2 copies/μL, which was more sensitive than the conventional RT-PCR (10 3 copies/μL). Likewise, Ma et al [25] recently reported a detection limit as low as 10 2 copies/μL of PDCoV by a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay, whose sensitivity was the same as that of LFD-RPA developed in the present study. The specific validation test indicated that the method is useful for PDCoV detection, and there was no cross-reaction with the several reference swine viruses used in the study.…”

“…The top line on the lateral flow dipstick is the control line, and the next line is the test line the reaction tube [26]. Real-time RT-PCR and RT-PCR, which are simple methods for detecting PDCoV, have been developed with high sensitivity and specificity, but these assays are difficult to perform in resource-poor areas and are not appropriate for field detection of PDCoV [25,30]. Recently, a rapid and accurate real-time RT-RPA was established for the detection of PDCoV [25], while observations of results require specific equipment.…”

“…Real-time RT-PCR and RT-PCR, which are simple methods for detecting PDCoV, have been developed with high sensitivity and specificity, but these assays are difficult to perform in resource-poor areas and are not appropriate for field detection of PDCoV [25,30]. Recently, a rapid and accurate real-time RT-RPA was established for the detection of PDCoV [25], while observations of results require specific equipment. However, compared with previous studies, LFD-RPA was carried out at a lower temperature (10-37°C), under a shorter time (< 20 min) and required only a pair of primers in this study.…”

Establishment of a Real-Time Recombinase Polymerase Amplification Assay for the Detection of Avian Reovirus

“…RPA which was invented in 2006 has been experiencing rapid development (19,27). The RPA method was simpler and faster compared to other detection methods.…”

Tandem Mass Tag-Based Quantitative Proteome Analysis of Porcine Deltacoronavirus (PDCoV)-Infected LLC Porcine Kidney Cells

“… 1 The viral genome is approximately 25.4 kb in length, encoding four nonstructural proteins (replicase 1a/b, NS6, and NS7) and four major structural proteins (spike (S), envelope (E), membrane (M), and nucleocapsid (N)). 6 So far, although PDCoV can be accurately and rapidly detected in the laboratory, 7 knowledge of its pathogenesis and an effective vaccine are still insufficient, and PDCoV has caused substantial economic losses to the swine industry worldwide.…”