Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey.

By the end of 2004, the Canadian swine population had experienced a severe 2 increase in the incidence of Porcine circovirus-associated disease (PCVAD), a problem that was 3 associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously 4 unrecovered in North America. Thus it became important to develop a diagnostic tool that could 5 differentiate between the old and new circulating genotypes (PCV-2a and -2b, respectively). 6 Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that 7 could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A 8 retrospective epidemiological survey that used the mrtqPCR assay was performed to determine if 9 cofactors could affect the risk of PCVAD. From 121 PCV-2–positive cases gathered for this 10 study, 4.13%, 92.56% and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, 11 respectively. In a data analysis using univariate logistic regressions, PCVAD compatible 12 (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and 13 respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 14 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that 15 PCVAD/c score was affected by PCV-2 viral load (P = 0.0161) and IHC (P = 0.0128), but not by 16 the PRRSV variables (P > 0.9); suggesting that mrtqPCR in tissue is a reliable alternative to IHC. 17 Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major 18 swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus 19 suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical 20 diseases.