Development and Clinical Applications of a 5-Plex Real-Time RT-PCR for Swine Enteric Coronaviruses

Abstract

A PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex real-time RT-PCR was developed and validated for the simultaneous detection and differentiation of four swine enteric coronaviruses (PEDV, PDCoV, TGEV and SADS-CoV) in one PCR reaction (XIPC serves as an exogenous internal positive control). The 5-plex PCR had excellent analytical specificity, analytical sensitivity, and repeatability based on the testing of various viral and bacterial pathogens, serial dilutions of virus isolates, and in vitro transcribed RNAs. The 5-plex PCR had comparable diagnostic performance to a commercial PEDV/TGEV/PDCoV reference PCR, based on the testing of 219 clinical samples. Subsequently, 1807 clinical samples collected from various U.S. states during 2019–2021 were tested by the 5-plex PCR to investigate the presence of SADS-CoV in U.S. swine and the frequency of detecting swine enteric CoVs. All 1807 samples tested negative for SADS-CoV. Among the samples positive for swine enteric CoVs, there was a low frequency of detecting TGEV, an intermediate frequency of detecting PDCoV, and a high frequency of detecting PEDV. Although there is no evidence of SADS-CoV presence in the U.S. at present, the availability of the 5-plex PCR will enable us to conduct ongoing surveillance to detect and differentiate these viruses in swine samples and other host species samples as some of these coronaviruses can cause cross-species infection.

Isolation and Pathogenicity Analysis of a G5P[23] Porcine Rotavirus Strain

“…On 6 January 2023, 28 diarrheal samples were collected from piglets in a pig farm infected by PoRV in Jiangsu Province, China. The virus isolated from these samples was identified as PoRV through Reverse Transcription-Polymerase Chain Reaction (RT–PCR) and Quantitative real-time polymerase chain reaction (qRT–PCR) using VP6 ( Supplementary Table S1 ) [ 22 , 23 , 24 ]. Samples of the intestinal contents were homogenized in serum-free Dulbecco’s modified Eagle medium [DMEM] (Invitrogen, Carlsbad, CA, USA) containing 1% penicillin–streptomycin [10,000 units/mL penicillin and 10,000 µg/mL streptomycin] (Gibco™, New York, MT, USA) and 0.3% trypsin phosphate broth (Sigma-Aldrich, St. Louis, MO, USA).…”

Porcine Respiratory Coronavirus (PRCV): Isolation and Characterization of a Variant PRCV from USA Pigs

“…The XIPC sequence was not present in any analyzed pathogens or host species, and it was a fragment of nucleotides that was artificially designed and synthesized with a T7 promoter at the 5′ upstream. XIPC DNA was in vitro transcribed into XIPC RNA, which was added to the extraction lysis buffer before nucleic acid extraction [ 34 ].…”

“…Subsequently, the analytical sensitivity of the 3-plex PCR was determined using serial dilutions of in vitro transcribed (IVT) RNAs of PRCV and TGEV, with 3 replicates at high concentrations and 20 replicates at low concentrations for each dilution. The IVT RNAs were generated from the PRCV gBlock DNA fragment (1127 nucleotides in length) containing the partial PRCV nucleocapsid gene and the TGEV gBlock DNA fragment (1138 nucleotides in length) containing the partial TGEV spike gene, respectively, following the previously described procedures [ 34 ].…”

Current State of Molecular and Serological Methods for Detection of Porcine Epidemic Diarrhea Virus

“…The presence of multiple primer pairs in multiplex PCR assays may result in the formation of nonspecific products. Therefore, special attention should be paid to designing specific primers and probes [ 84 ]. The primer length, melting temperature, different annealing temperatures for different primers, guanine–cytosine content, secondary structure, repeats, 3′ end stability, product position and optimization of PCR conditions have to be considered.…”