Evaluation of a Nested Polymerase Chain Reaction Assay to Differentiate Between Two Genotypes of Porcine Circovirus-2

Abstract

Porcine circovirus-2 (PCV-2) is associated with several diseases in pigs, including postweaning multisystemic wasting syndrome (PMWS). A new genotype of PCV-2 was isolated from swine farms with and without clinical PMWS in North America. The new genotype was differentiated in a separate cluster by phylogenetic analyses and is now named PCV-2b compared with PCV-2a for the previously known genotype. The purpose of this study was to develop and evaluate a nested polymerase chain reaction (nPCR) assay to detect and differentiate between PCV-2a and PCV-2b. Genotype-specific primer sets were designed by using sequence data published for different PCV-2 strains. Specificity and sensitivity of the nPCR were examined by using PCV-2 isolates with known genotype. Nested PCR was found to be highly specific and sensitive for detecting and differentiating between the PCV-2 genotypes compared with the conventional 1-step PCR assay. Nested PCR was applied to detect PCV-2 and to identify the genotype in serum samples from swine farms with and without a clinical history of PMWS. Of 60 serum samples collected from 4 farms during clinical PMWS outbreaks, PCV-2a and PCV-2b were detected in 6 and 49 samples, respectively. Six of the 10 samples from one of the 4 farms had both PCV-2a and PCV-2b. Of 20 serum samples from 2 farms without PMWS, 11 were positive for PCV-2a only. These results suggest that the differential nPCR can be used to detect PCV-2 and to differentiate the 2 genotypes from field samples.

Development of a loop-mediated isothermal amplification method to rapidly detect porcine circovirus genotypes 2a and 2b

“…Generally, the nucleotide sequencing has been used to detect PCV2a and PCV2b [12], other approaches included PCR [16], nested PCR [17], PCR-RFLP [18] and TaqMan-based assay [27]. However, these methods were usually time consuming and high technical requirement, and were difficult to popularize in veterinary clinic practice.…”

“…These controversial PCV2 new genotypes need further investigation. At present, PCR [16], nested PCR [17], PCR-RFLP [18], TaqMan-based assay [19], nucleotide sequencing and phylogenetic analysis [12] have been used to detect and differentiate PCV2a and PCV2b. However, the application of these methods is limited somewhat by the expensive PCR equipment, the long detection period or the high technical requirements [20,21].…”

Genotypic Change and Phylogenetic Analysis of Porcine Circovirus Type 2 in Taiwanese Pig Herds

“…The PCV2c genotype has only been found in Denmark to date [31]. In recent years, several reports have suggested that PCV2b is the predominant genotype in many countries worldwide, including Brazil [8,11], Canada [14,15], China [17,21,38], Cuba [29], Denmark [12], Germany [30], Japan [32], Korea [1,2,19,20], Slovakia [37], Spain [10], Sweden [33], Switzerland [39], Thailand [18] and the U.S.A. [6,23]. PCV2b might be more pathogenic than PCV2a [16,32].…”

Retrospective Study of Porcine Circovirus Type 2a and 2b Between 1999 and 2016 in Taiwan

“…Recently, there are several excellent methods that can be used to identify the PCV2a and PCV2b; these include polymerase chain reaction (PCR) assay, nested PCR, real-time PCR and restriction fragment length polymorphism (RFLP) [5,14,15,16]. These methods are convenient and allow the testing of large number of clinical samples.…”