Multiplex Real-Time RT-PCR Assays for Detection and Differentiation of Porcine Enteric Coronaviruses

Abstract

It is important to be able to detect and differentiate between distinct porcine enteric coronaviruses that can cause similar diseases. However, the existence of naturally occurring recombinant coronaviruses such as swine enteric coronavirus (SeCoV) can give misleading results with currently used diagnostic methods. Therefore, we have developed and validated three duplex real-time quantitative RT-PCR assays for the simultaneous detection of, and differentiation between, porcine epidemic diarrhea virus (PEDV) and SeCoV. Transmissible gastroenteritis virus (TGEV) is also detected by two out of these three assays. In addition, a novel triplex assay was set up that was able to detect and differentiate between these alphacoronaviruses and the porcine deltacoronavirus (PDCoV). The validated assays have low limits of detection, close to 100% efficiency, and were able to correctly identify the presence of PEDV and SeCoV in 55 field samples, whereas 20 samples of other pathogens did not give a positive result. Implementing one or more of these multiplex assays into the routine diagnostic surveillance for PEDV will ensure that the presence of SeCoV, TGEV, and PDCoV will not go unnoticed.

Establishment and Application of a Quadruplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, Porcine Circovirus Type 3, and Streptococcus suis

“…Recently, Lung et al reported a novel automated microarray prototype for streamlining post-PCR processing for simultaneous detection and genotyping of bacteria ( Mycoplasma hyopneumonia , A. pleuropneumoniae, Salmonella choleraesuis , and S. suis ) and viruses (PRRSV, SIV, PCV2, PRCV) [ 27 ]. Real-time PCR’s high specificity and sensitivity have proven advantageous in swift pathogen detection [ 28 ]. However, a multiplex real-time PCR approach for simultaneous detection of PRRSV, PCV2, PCV3, and SS pathogens in pigs is yet to be reported.…”

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A

“…Currently, the commonly used molecular detection techniques for PEDV, TGEV and PoRVA include RT-PCR, nested RT-PCR, qRT-PCR, reverse transcription loop-mediated isothermal amplification, reverse transcription recombinase-aided amplification, and CRISPR-Cas nucleic acid detection (Marthaler et al, 2014;Areekit et al, 2022;Wu et al, 2022;Lazov et al, 2023;Xia et al, 2024). RT-PCR and nested RT-PCR are not capable of quantitative analysis, and their operation is cumbersome, time-consuming and less sensitive.…”