Abstract
BackgroundPorcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. Since then, PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. Therefore, it is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV.ResultsIn the present study, a highly sensitive and specific diagnostic method using recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed for rapid and visual detection of PDCoV. The system can be performed under a broad range of temperature conditions from 10 to 37 °C, and the detection of PDCoV can be completed in 10 min at 37 °C. The sensitivity of this assay was 10 times higher than that of conventional PCR with a lower detection limit of 1 × 102 copies/µl of PDCoV. Meanwhile, the LFD-RPA assay specifically amplified PDCoV, while there was no cross-amplification with other swine-associated viruses, including Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine kobuvirus (PKoV), Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2), Classical swine fever virus (CSFV) and Seneca valley virus (SVV). The repeatability of the test results indicated that this assay had good repeatability. In addition, 68 clinical samples (48 fecal swab specimens and 20 intestinal specimens) were further tested by LFD-RPA and RT-PCR assay. The positive rate of LFD-RPA clinical samples was 26.47% higher than that of conventional PCR (23.53%).ConclusionsThe LFD-RPA assay successfully detected PDCoV in less than 20 min in this study, providing a potentially valuable tool to improve molecular detection for PDCoV and to monitor the outbreak of PDCoV, especially in low-resource areas and laboratories.
Highlights
Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China
Optimization of LFD-Recombinase polymerase amplification (RPA) conditions In the detection of PDCoV by the LFD-RPA assay, an intense test band could be observed on the lateral flow dipstick with the specific primers and probe
The results demonstrated that the limit of detection with the LFD-RPA was 102 copies/μL, which was higher than that of conventional PCR (103 copies/μL) (Fig. 2a and b)
Summary
Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. It is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV. Porcine Deltacoronavirus (PDCoV) is an enveloped, single-stranded, positive-sense RNA virus and a member of the genus Deltacoronavirus in the family Coronaviridae that causes diarrhea, vomiting, dehydration and mortality in neonatal piglets [1,2,3]. PDCoV infection has become prevalent in pig farms around the world, which has caused enormous economic losses in multiple countries and remains a serious challenge to the swine industry [12, 13]. It is essential to develop a rapid, simple and highly sensitive diagnostic method to improve the efficacy of current control programs
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