Development and application of a novel Bio–Plex suspension array system for high–throughput multiplexed nucleic acid detection of seven respiratory and reproductive pathogens in swine

Development of a real‐time PCR assay for detection of African swine fever virus with an endogenous internal control

“…Another advantage of the ASFV multiplex assay we developed is that it is more sensitive as compared to the singleplex Zsak assay (Zsak et al., 2005). Based on the analysis of standard plasmid, the ASFV multiplex assay showed higher sensitivity (6 copies per reaction) compared to other ASFV real‐time PCR assays (Fernandez‐Pinero et al., 2013; Haines et al., 2013; McKillen et al., 2010; Wang, Jia, et al, 2019; Zsak et al., 2005), LAMP assays (James et al., 2010; Wang, Yu, et al, 2019), RPA assays (Wang et al., 2017) or regular PCR assays (Gonzague, Plin, Bakkali‐Kassimi, Boutrouille, & Cruciere, 2002; Luo et al., 2017; Xiao et al., 2018). Although the LODs of the ASFV multiplex assay and Zsak assay are the same using cell culture isolates, our new assay is 3‐Ct lower than the Zsak assay, indicating it is analytically a more sensitive and a more reliable assay.…”

“…Compared to other molecular diagnostic methods, such as loop‐mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), bead‐based multiplex PCR and whole genome sequencing, the real‐time PCR assay is considered more reliable, sensitive and cost effective (O'Donnell et al., 2019; Wang, Yu, et al, 2019; Wang, Wang, Geng, & Yuan, 2017; Xiao et al., 2018). Although several ASFV real‐time PCR assays were developed recently (Fernandez‐Pinero et al., 2013; Haines, Hofmann, King, Drew, & Crooke, 2013; McKillen et al., 2010; Wang, Jia, et al, 2019), their strain coverage and detection sensitivity can be further improved, and an internal control should be included to ensure detection accuracy.…”

“…Compared to other molecular diagnostic methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), bead-based multiplex PCR and whole genome sequencing, the real-time PCR assay is considered more reliable, sensitive and cost effective (O'Donnell et al, 2019;Wang, Yu, et al, 2019;Wang, Wang, Geng, & Yuan, 2017;Xiao et al, 2018).…”

Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

“…Multiplex RT-PCR assays for rapid detection and genotyping of CSFVs [ 36 , 37 ], simultaneous detection, and differentiation of common swine viruses [ 38 , 39 , 40 , 41 ] have been developed. Additionally, multiplex combined high-throughput molecular diagnostic platform, user-friendly electronic microarray, magnetoelastic sensor, and microfluidic detection systems were developed as potential alternatives for detection and surveillance of CSFV infection [ 42 , 43 , 44 , 45 , 46 ]. These assays can save considerable time and effort without compromising robustness and sensitivity and can reduce the sample and reagent requirement as well.…”

Development of a bead‐based assay for detection and differentiation of field strains and four vaccine strains of type 2 porcine reproductive and respiratory syndrome virus (PRRSV‐2) in the USA

“…Simultaneous detection and differentiation of PRRSV‐1 and PRRSV‐2 by real‐time PCR has been more frequently used (Chen et al., 2019; Park et al., 2019). Some other technologies are also applied for PRRSV detection, including full‐genome sequencing (Tan, Dvorak, & Murtaugh, 2019), restriction fragment length polymorphism (RFLP) analysis of ORF5 sequences (Ramirez et al., 2019), loop‐mediated isothermal amplification (LAMP) (Park et al., 2016), melting curve analysis (Sun et al., 2018), microarray (Erickson et al., 2018), droplet digital PCR (Yang, Xi, et al, 2017), next generation sequencing (NGS) (Zhang et al., 2017) and Luminex xTAG (Xiao et al., 2018) which allowed simultaneous detection of 7 respiratory and reproductive pathogens. However, an assay for detecting PRRSV and distinguishing field strains from four commercial vaccine strains (Ingelvac PRRS MLV, Ingelvac PRRS ATP, Fostera PRRS, and Prime Pac PRRS) has not been described.…”