Comparison of Multiplex Real-Time PCR and PCR-Reverse Blot Hybridization Assays for the Direct and Rapid Detection of Porcine Circovirus Type 2 Genotypes

Abstract

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), poses a serious economic threat for the swine industry. Currently, PCV2 is classified into five major genotypes: PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. The aim of this study is to evaluate the performance of two commercially available methods, multiplex real-time PCR assay and PCR-reverse blot hybridization assay (REBA), for the rapid detection of PCV2 and direct identification of PCV2 genotypes from clinical samples as well as to compare the results with that of sequence analysis. Molecular diagnostic methods were used to evaluate a total of 180 samples, including tissues and blood samples from pigs that were suspected of PCVAD infection. The results of this study showed that the detection rate for positive PCV2 was 48.3% (n = 87) in both multiplex real-time PCR and PCR-REBA methods. Using sequence analysis, which is the gold standard, and multiplex real time PCR assay, the sensitivity, specificity, positive predictive value, and negative predictive value of PCV2 genotyping were found to be 97.1% (n = 67, 95% CI 0.894-0.998, p < 0.001), 100% (n = 93, 95% CI 0.966-1.000, p < 0.001), 100% (95% CI 0.953-1.000, p < 0.001), 97.9% (95% CI 0.921-0.998, p < 0.001), respectively. The results of PCR-REBA were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). The most prevalent genotypes detected in this study were PCV2d (n = 53, 60.9%), followed by PCV2a (n = 17, 19.5%), PCV2b (n = 14, 16.1%), and PCV2a/b co-infection (n = 3, 3.5%). Both the methods required ∼3 h for completion. Therefore, we conclude that two molecular methods are rapid and reliable for the characterization of the causative pathogen with PCV2 genotypes.

Development of Dry and Liquid Duplex Reagent Mix-Based Polymerase Chain Reaction Assays as Novel Tools for the Rapid and Easy Quantification of Bovine Leukemia Virus (BLV) Proviral Loads

“…The TBIRD qPCR master mix, which has high sensitivity, specificity, and cost-effectiveness, is usually used for the Single-CoCoMo assay [10,11,29,31,33,34,[41][42][43][44][45]. In addition, the TBIRD qPCR master mix has been used in several multiplex qPCR assays [46]. Contrary to our expectations, in this study, the use of TBIRD in the Liquid Dual-CoCoMo assay showed reduced measurement accuracy compared with the GeneAce-based Dual CoCoMo assay.…”

Implementation of point-of-care platforms for rapid detection of porcine circovirus type 2

“…To establish prevention and control strategies for PCV2, PCR assays are sensitive methods to detect viruses in animals [ 28 ]. Several different PCR-related methods have been employed, such as ddPCR [ 17 ], real-time PCR [ 12 14 18 ], and LAMP [ 15 16 ]. The ddPCR require an expensive thermocycler and are time-consuming and difficult to perform for inexperienced investigators.…”

“…PCV2 was primarily determined by the restriction fragment length polymorphism (RFLP) assay for genotype differentiation [ 10 ] or detected viral DNA in tissues through immunohistochemistry (IHC) [ 11 12 ] and/or in situ hybridization (ISH) [ 13 ]. PCR-based diagnostic methods were also proposed, including real-time PCR [ 12 ], multiplex real-time PCR [ 14 ], loop-mediated isothermal amplification (LAMP) PCR [ 15 16 ], droplet digital PCR (ddPCR) [ 17 ], and SYBR TaqMan-based PCR [ 18 ] assays. Furthermore, commercial kits or antigens detecting PCV2 antibodies based on enzyme-linked immunosorbent assay (ELISA) were also available [ 19 20 21 ].…”

Revisiting Porcine Circovirus Infection: Recent Insights and Its Significance in the Piggery Sector

“…The TaqMan-based quadruplex real-time PCR was able to differentiate four species of PCV strains in clinical samples, rapidly and simultaneously [ 228 ]. Recently, multiplex real-time PCR has been developed, which is rapid, sensitive, efficient, and highly specific, allowing the detection and discrimination of different PCVs from clinical samples and even comparison of different genotypes of a particular species without requiring any specialized laboratory equipment [ 229 , 230 ]. Droplet digital PCR (ddPCR) is another novel PCR technology used for the detection of PCV2 and PCV3 with greater analytical sensitivity than TaqMan real-time PCR [ 231 , 232 ].…”