Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus

Abstract

Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect the two viruses and differentiate PEDV from PDCoV.

Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014

“…Some groups have recently reported the emergence of PDCoV in different areas of the Chinese mainland, and interestingly, we detected PDCoV in pig diarrhea samples collected as early as 2004 (Chen et al, 2015a(Chen et al, , 2015bDong et al, 2015;Song et al, 2015;Wang et al, 2015aWang et al, , 2015b. With increased concern about this newly emerging enteric disease, reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, nested RT-PCR, and singleplex RT insulated isothermal PCR methods have been developed to detect PDCoV in clinical samples Wang et al, 2014a;Song et al, 2015;Zhang et al, 2015Zhang et al, , 2016. Enzyme-linked immunosorbent assays for PDCoV-specific serum antibodies, based on the PDCoV S1 or nucleocapsid protein, have also been used to determine the prevalence of anti-PDCoV immunoglobulin G (IgG) in pig serum (Su et al, 2015;Thachil et al, 2015).…”

Porcine deltacoronavirus: Overview of infection dynamics, diagnostic methods, prevalence and genetic evolution

“…Due to two steps of reaction set up, nested RT-PCR is more prone to contamination and special attention needs to be paid when performing nested RT-PCR. The standard RT-PCR assays especially the nested RT-PCR assays have not been widely used by (Chen et al, 2015b;Ma et al, 2015;Marthaler et al, 2014b;Zhang et al, 2016). These rRT-PCR assays target the conserved regions of PDCoV M or N genes with the limit of detection (LOD) of 2 viral RNA copies per reaction for the assay described by Marthaler et al (2014b) and LOD of 0.056 TCID 50 per reaction for the assay described by Chen et al (2015b) .…”

Porcine Coronaviruses

“…Diagnosis can also be made utilising RT-PCR assays that target a conserved region of PDCoV M or N genes (Marthaler et al 2014b;Wang et al 2014c), IF or IHC using virus-specific MAbs or polyclonal antibodies Jung et al 2015b;Ma et al 2015), and in situ hybridisation (Jung et al 2015b). A duplex real-time RT-PCR assay for detection of PDCoV and/or differentiation from PEDV in intestines and faeces was developed (Zhang et al 2016b).…”