A novel swine acute diarrhea syndrome Coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in southern China in 2017. A simple and rapid detection test was developed for this virus using real-time RT-LAMP based on the conserved N gene of the virus. The method had a detection limit of 1.0 × 10 copies/μL with no cross-reactions with classical swine fever virus, porcine and respiratory syndrome virus NA, porcine and respiratory syndrome virus EU, transmissible gastroenteritis coronavirus, foot and mouth disease virus, porcine epidemic diarrhea virus (S-INDEL and non-S-INDEL), swine influenza virus subtype H1N1, porcine circovirus type 2, seneca valley virus, porcine parvovirus, porcine deltacoronavirus and rotavirus. This method was also reproducible. Twenty of 24 clinical samples were identified as SADS-CoV RNA-positive by the real-time RT-LAMP and the results were consistent with that of the real time RT-PCR method. This new method for detecting SADS-CoV is specific and sensitive for the detection of SADS-CoV.

BackgroundMurine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV.ResultsThe detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA.ConclusionsA broadly reactive RT-RPA assay was successfully established for MNV detection.

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.

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

A B S T R A C TSwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus which was associated with severe diarrhea disease in pigs. SADS-CoV was first detected and identified as the causative agent of a devastating swine disease outbreak in southern China in 2017. Routine monitoring and early detection of the source of infection is therefore integral to the prevention and control of SADS-CoV infection. In this study, a SYBR greenbased real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique was established for rapid detection and monitoring of this emerging virus. Specific primers were designed based on the conserved region within the M gene of the viral genome. The lowest detection limit of the RT-qPCR assay was 10 copies/μL. This assay was specific and had no cross-reaction with other 11 swine viruses. The positive rate of 84 clinical samples for the SYBR green-based RT-qPCR and the conventional RT-PCR was 73.81% (62/84) and 53.57% (45/84), respectively. These results demonstrated that the SYBR green-based RT-qPCR technique was an effectively diagnostic method with higher sensitivity than probe-based RT-qPCR and gel-based RT-PCR for detection and epidemiological investigations of SADS-CoV. ⁎ Corresponding authors.

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