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

Joong Ki Song,Hyunil Kim,Seongho Shin,Hye-Young Wang

doi:10.3389/fvets.2020.00200

Joong Ki Song, Hyunil Kim + Show 2 more

Open Access

https://doi.org/10.3389/fvets.2020.00200

Copy DOI

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.

Highlights

Different from the non-pathogenic porcine circovirus (PCV) type 1 strain [1], PCV type 2 is considered to be an important emerging pathogen that causes porcine circovirus associated diseases (PCVAD) including postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), enteritis, Identification of Porcine circovirus type 2 (PCV2) Genotypes reproductive failure [2, 3], and one of the most economically important swine diseases worldwide [4]
We evaluated the clinical applicability of multiplex real-time PCR and PCR-reverse blot hybridization assay (REBA) methods and compared their efficiency to that of the sequence analysis method used for detecting PCV2 and differentiating the different PCV2a-e genotypes directly from the serum and tissue samples of pigs
To evaluate the diagnostic performance of the multiplex realtime PCR and PCR-REBA methods, a total of 180 samples suspected to be infected with PCVAD including 109 tissues and 71 bloods were provided by the Optipharm Animal Disease Diagnostic Center, which was commissioned from January to December, 2019

Summary

Introduction

Different from the non-pathogenic porcine circovirus (PCV) type 1 strain [1], PCV type 2 is considered to be an important emerging pathogen that causes porcine circovirus associated diseases (PCVAD) including postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), enteritis, Identification of PCV2 Genotypes reproductive failure [2, 3], and one of the most economically important swine diseases worldwide [4]. Rep is a non-structural protein and is responsible for the viral replication, while the structural Cap protein controls the immunogenicity of the virus [10,11,12,13]. PCV2 has undergone much genetic variation in recent years and has been divided into five genotypes, namely PCV2ae strains, which are classified based on the diversity level of the ORF2 nucleotide sequences [14, 15]. It has been demonstrated experimentally that subclinical PCV2 infection might be associated with decreased vaccine efficacy [18]. PCV2 subclinical infection is the most common form of infection in pigs but is resistant to the effect of vaccines. Rapid and early identification of PCV2 subclinical infection is very important for the effective prophylaxis against PCVAD [19]

Objectives

Methods

Results

Discussion

Conclusion