Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses

Abstract

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based realtime PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 10 2 copies/μL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

Melatonin and other indoles show antiviral activities against swine coronaviruses in vitro at pharmacological concentrations

“…including porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV), are prevalent in pig populations and cause huge economic losses. [4][5][6][7] Porcine enteric coronaviruses have similar epidemiological, clinical, and pathological characteristics. Infection with TGEV and PEDV is characterized by severe diarrhea, vomiting, and dehydration, leading to high morbidity and mortality in piglets.…”

Development and Application of a Reverse-Transcription Recombinase-Aided Amplification Assay for Porcine Epidemic Diarrhea Virus

“…We found that Jiang established an RT-PCR sensitivity of 10 4 copies/µL [ 73 ], and Pan established an RT-qPCR sensitivity of 10 2 copies/µL [ 74 ]. In this study, the sensitivity of the RT-RAA was 10 1 copies/µL, which was 1000 times for RT-PCR and 10 times for RT-qPCR.…”

Development of a One-Step Multiplex Real-Time PCR Assay for the Detection of Viral Pathogens Associated With the Bovine Respiratory Disease Complex

“…To improve the amplification efficiency of different primers and probes, the annealing temperature of the primers was set at 57 • C, the annealing temperature of the probe was set at 67 • C. Notably, in this developed assay, the primer/probe set of BVDV showed the lowest amplification. One possible explanation is that the fluorescence of Cy5 was the weakest and very susceptible to interference from other fluorophores due to its own physical properties (30). Despite the optimization such as primer/probe concentration and annealing temperature, the improvement effect was not significant.…”

“…Conventional RT-PCR and PCR have been widely applied for pathogen detection. However, this method is only suitable for infection by a single pathogen, and time-consuming ( 29 , 30 ). As bovine respiratory disease syndrome is mainly caused by co-infection of multiple pathogens, it is urgent to develop a rapid and convenient detection method to simultaneously the pathogens causing BRDC.…”