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

Abstract

Background: Porcine 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.Results: In 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 × 10 2 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%). Conclusions: The 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.

One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

“…In recent years, RPA combined with visual LFS has been widely developed and employed to detect pathogens ( Liu et al, 2018 ; Sun et al, 2019 ; Gao et al, 2020 ; Yang et al, 2020b ). To detect RNA viruses, an additional reverse transcription step is required before RPA-LFS.…”

“…To detect RNA viruses, an additional reverse transcription step is required before RPA-LFS. To the best of our knowledge, reverse transcription and RPA were performed in different tubes in most previous reports ( Sun et al, 2019 ; Gao et al, 2020 ; Yang et al, 2020b ). However, some studies attempted to integrate reverse transcription with RPA ( Liu et al, 2018 ; Sun et al, 2018 ).…”

Point of care diagnostics and non-invasive sampling strategy: a review on major advances in veterinary diagnostics

“…Detection of the H9-subtype of Avian influenza virus by RPA LFD assay with 10 × more sensitivity than conventional RT-PCR has also been documented (Wang et al 2019). Several other significant animal diseases for which RPA-LFD assays have been developed include detection of Porcine circovirus type 2 (PCV-2) (Yang et al 2017b), PPRV (Yang et al 2017c), ASFV (Miao et al 2019), Porcine deltacoronavirus (PDCoV) (Gao et al 2020), Pasteurella multocida (Zhao et al 2019), Bovine viral diarrhoea virus (BVDV) (Hou et al 2018a), Bovine ephemeral fever virus (BEFV) (Hou et al 2018b), Mycoplasma bovis (Zhao et al 2018), Infectious bovine rhinotracheitis virus (IBRV) (Hou et al 2017), FMDV (Wang et al 2018b), Brucella species (Qin et al 2019), PRRSV (Wang et al 2017) and LSDV (Shalaby et al 2016). The use of RPA-LFD assay in diagnostic application is therefore becoming a molecular tool of choice for the rapid, specific, and cost-effective approach for identification of animal diseases in laboratory settings as well as in field conditions.…”

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick

“…Recombinase polymerase amplification (RPA), which was first reported in 2006, is an isothermal amplification technique [ 20 ] that does not require thermal denaturation of the template and can be operated at a low and constant temperature in a simple water bath, or even at body temperature. To date, RPA-based detection has been successfully applied for the detection of severe acute respiratory syndrome coronavirus 2, classical swine fever virus (CSFV), African swine fever virus, porcine circovirus 2 (PCV2), foot-and-mouth disease virus, porcine deltacoronavirus, and other viruses [ 21 – 26 ].…”