A multiplex real-time PCR assay for the detection and differentiation of the newly emerged porcine circovirus type 3 and continuously evolving type 2 strains in the United States

Abstract

A B S T R A C TA multiplex quantitative real-time polymerase chain reaction (mqPCR) assay was developed and validated for the detection and differentiation of porcine circovirus type 3 (PCV3) and type 2 (PCV2) strains. The assay coverage was 97.9% (184/188) for PCV3 and 99.1% (1889/1907) for PCV2 sequences that were available from the current GenBank database. The PCR amplification efficiencies were 98-99% for plasmids, and 92-96% for diagnostic samples, with correlation coefficients all greater than 0.99. The limit of detection (LOD) determined as plasmid copies per reaction was 17 for PCV3 and 14 for PCV2. The assay specifically detected the targeted viruses without cross reacting to each other or to other common porcine viruses. Among 336 swine clinical samples collected in 2018, 101 (30.1%) were PCV3 positive, 56 (16.7%) were PCV2 positive and 18 (5.4%) were co-positives. Sixty selected PCV3 positives were confirmed by Sanger sequencing, and 53 of the 56 PCV2 positive samples were tested positive by another validated PCR assay.

WITHDRAWN: Identification of Porcine circovirus type (PCV2) and type 3 (PCV3), Porcine 2 parvovirus (PPV) in swine by multiplex PCR test

“…However, those PCRs are designed for detection of a single viral agent at a time. Recently, duplex PCRs were developed for simultaneous detection of both PCV2 and PCV3 [26,27,28]. In the present study, we report the development and validation of a mPCR that allows simultaneous detection of these three important viruses in clinical samples.…”

Development of a differential multiplex real-time PCR assay for porcine circovirus type 2 (PCV2) genotypes PCV2a, PCV2b and PCV2d

“…Therefore, when single nucleotide mismatch in such situations is considered in the design of the primers and probes, strain coverage can potentially increase to 93.4 %–95.1 %. With that said, this genotyping assay should not replace general detection assays that may have higher strain coverage ( Wang et al, 2019a ).…”

“…This simplified method is a low-cost and faster turnaround protocol, and should facilitate the genotyping process for field PCV2 strains. We have already developed a PCV2 general assay using the conserved ORF1 gene as the target ( Wang et al, 2019a ), which has enabled us to have a much higher strain coverage (99.1 %) for PCV2 detections. By utilizing that generic test for general detection and surveillance, followed by the genotyping PCR reported here, we should be able to detect and differentiate the majority of field PCV2 strains.…”

“…Porcine circovirus type 2 (PCV2) plays a significant role in porcine circovirus associated diseases (PCVAD) and is one of the most economically important porcine viral pathogens ( Gillespie et al, 2009 ). Since it was first isolated as the causative agent of postweaning multisystemic wasting syndrome in 1998 ( Ellis et al, 1998 ), PCV2 has been a highly prevalent disease worldwide, including in Asia, America and Europe ( Saporiti et al, 2020 ; Wang et al, 2019a ; Zheng et al, 2020 ).…”

Advanced target‐specific probe‐based real‐time loop‐mediated isothermal amplification assay for the rapid and specific detection of porcine circovirus 3

“…In this study, a more rapid and specific rLAMP assay using targetspecific fluorescence assimilating probes to facilitate real-time detection of PCV3 was successfully developed and was evaluated with clinical samples. To the best of our knowledge, the PCV3 rLAMP assay described herein is the first assimilating probe-applied LAMP assay used to diagnose PCV3 in real time; this method is more specific and reliable than the conventional LAMP assay that relies on non-target-specific monitoring methods (Park et al, 2018;Wang et al, 2019). As a result of the clinical evaluation, it was confirmed that the developed rLAMP has similar sensitivity and specificity to the previously reported qPCR for detection of PCV3 (Table 2 and each method has its unique pros and cons (Gadkar, Goldfarb, Gantt, & Tilley, 2018;Kouguchi, Fujiwara, Teramoto, & Kuramoto, 2010;Kubota et al, 2011;Kubota & Jenkins, 2015;Liu et al, 2017;Nyan & Swinson, 2015;Tanner, Zhang, & Evans, 2012;Wang et al, 2017).…”