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

Abstract

Porcine circovirus type 3 (PCV3) is an emerging viral pathogen that has been identified in pigs with various clinical signs. For rapid and specific detection of PCV3, an advanced real-time loop-mediated isothermal amplification (rLAMP) assay that uses both assimilating probes and swarm primers were developed and evaluated in this study. The assay specifically amplified PCV3 DNA, but it did not amplify other porcine viral nucleic acids. The limit of detection of rLAMP with swarm primers was 50 PCV3 DNA copies/reaction, which was comparable to that of the real-time quantitative polymerase chain reaction (qPCR) and 10 times more sensitive than rLAMP without swarm primers. In an evaluation of clinical samples, the rLAMP assay was able to detect PCV3 DNA within 17.34 ± 4.45 min, which is more rapid than what has been previously reported for the standard qPCR assay (31.78 ± 4.60 min). Detection with rLAMP was largely in agreement with that of the qPCR with a kappa value (95% confidence interval) of 0.98 (0.95-1.00). Taken together, these results suggest that the rLAMP assay presented will be a valuable tool for rapid, specific and reliable diagnosis of PCV3 in clinical samples.

Establishment and Application of Dual TaqMan Fluorescence Quantitative Polymerase Chain Reaction (PCR) for the Detection of Porcine Circovirus Types 2 and 3

“…Because of the difficulty of virus isolation and the lack of a commercial serological test kit, the detection of viral nucleic acids is presently the most effective diagnostic method for PCV detection. The main detection methods for PCV3 include conventional PCR, nested PCR, multiplex PCR, fluorescence quantitative PCR, and loop-mediated isothermal amplification (Ha et al 2018;Kim et al 2020;Wang et al 2019a;Zheng et al 2020). Several scholars have designed various primers for PCR detection according to the PCV3 gene, and established a rapid, specific, and sensitive PCR detection method and applied it in practice.…”

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection

“…Kubota et al developed the assimilating probe-based LAMP and subsequently designed a simple, low-power, portable handheld device to enable real-time detection of the LAMP amplicons [41,51]. This assay was mainly used for singleplex detection of viruses, and the sensitivities of this assay in the detection of foot-and-mouth disease virus (FMDV) and porcine circovirus 3 (PCV3) were 10 2 copies per µL and 50 copies per reaction, respectively [52,53].…”

Prevalence and co-infection status of three pathogenic porcine circoviruses (PCV2, PCV3, and PCV4) by a newly established triplex real-time polymerase chain reaction assay

“…In 2015, a third PCV species, PCV3, was discovered, in the USA, in pigs with PDNS, reproductive failure, myocarditis, and multisystemic inflammation (Phan et al, 2016;Palinski et al, 2017). Since then, PCV3 has been globally identified in pigs with various clinical symptoms consistent with those of PCVAD in several countries, including South America (Tochetto et al, 2018;Saraiva et al, 2019), Europe (Stadejek et al, 2017;Fux et al, 2018;Klaumann et al, 2019), and Asia, particularly China (Ku et al, 2017), and Korea (Kim et al, 2017;Kwon et al, 2017;Park et al, 2018;Kim et al, 2020). More recently, in 2019, a fourth novel and genetically distinct PCV, designated PCV4, was discovered in pigs with PDNS-like clinical syndrome in Hunan province, China (Zhang et al, 2020b).…”