Journal of Virological Methods 174 (2011) 53–59 Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Development and validation of a novel SYBR Green real-time RT-PCR assay for the detection of classical swine fever virus evaluated on different real-time PCR platforms Lester Josué Pérez a,1 , Heidy Díaz de Arce a,∗,1 , Joan Tarradas b , Rosa Rosell b,c , Carmen Laura Perera a , Marta Mu ˜ noz b , Maria T. Frías a , José Ignacio Nu ˜ nez b , Llilianne Ganges b a Centro Nacional de Sanidad Agropecuaria (CENSA), San Jose de las Lajas, La Habana, Apdo. 10, Cuba b Centre de Reserca in Sanitat Aimal (CReSA), UAB Bellaterra, 08193 Barcelona, Spain c Departament d’Agricultura, Alimentación i Acció Rural de la Generalitat de Catalunya (DAR), Spain Article history: Received 14 August 2010 Received in revised form 16 March 2011 Accepted 23 March 2011 Available online 31 March 2011 Keywords: Classical swine fever virus SYBR Green real-time RT-PCR Real-time PCR instrument abstract Classical swine fever is a highly contagious viral disease that causes significant economic losses in pig production on a global scale. The rapid dissemination of the virus and the variability of the clinical signs merit the development of swift and accurate classical swine fever virus (CSFV) detection methods, which can assist in disease control. The development and evaluation of a novel quantitative real-time RT-PCR assay for CSFV detection, based on SYBR Green coupled to melting curve analysis, is described. The ana- lytical and diagnostic performances of the method using two real-time PCR instruments were compared. The assay was specific and detected the major genotypes of CSFV. The limit of detection in cell culture medium and serum was 0.1 TCID50/reaction, while in tissue homogenate for both platforms, it was 1 TCID50/reaction. The limit of detection was 1, 10 and 10 2 gene copies/L when nuclease-free water, serum and tissue homogenate, respectively, were used as sample matrices for both instruments. The analysis of 108 tissue homogenate and serum samples from animals infected with CSFV naturally and experimentally and non-infected animals showed that the assay provided a highly sensitive and specific method for classical swine fever. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Classical swine fever (CSF) is a highly contagious viral disease of domestic pigs and wild boar (Moennig et al., 2003) that causes major losses in stock farming due to high mortality rates, compul- sory pig slaughter policies and bans on trade of live pigs and pig products (Terpstra and de Smit, 2000). CSF is a notifiable disease to the World Organization for Animal Health (OIE, 2008a). Classical swine fever virus (CSFV) is a member of the genus Pes- tivirus, which also includes Bovine viral diarrhoea virus 1 (BVDV-1), Bovine viral diarrhoea virus 2 (BVDV-2), Border disease virus (BDV) and a fifth tentative species, pestivirus of the giraffe (Thiel et al., 2005). The pestivirus genome consists of a single plus-strand RNA containing a single large open reading frame (ORF) flanked by two untranslated regions (UTRs). The ORF encodes a polyprotein of approximately 3900 amino acids, which is processed subsequently ∗ Corresponding author. Tel.: +53 47 863206; fax: +53 47 861104. E-mail addresses: heidydal@infomed.sld.cu, heidy@censa.edu.cu, heidydal@yahoo.es (H. Díaz de Arce). 1 Both authors contributed equally to the authorship of this work. by cellular and viral proteases into 12 mature proteins: four struc- tural (C, Erns, E1 and E2) and eight non-structural proteins (Npro, P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Meyers and Thiel, 1996). The rapid dissemination of the CSFV, as well as the variability of the clinical signs described among animals, compel the adoption of rapid and accurate methods as a basis for the implementation of effective control measures to prevent further spread of the disease (Moennig, 2000). The real-time RT-PCR method described below provides novel rapid means of virus detection in diagnostic labora- tories. The advantages of this approach over conventional RT-PCR methods include an enhanced sensitivity, a large dynamic range, a reduced risk of cross contamination, the possibility of scale up and the potential for an accurate target quantitation (Hoffmann et al., 2009). Recently, real-time RT-PCR assays based on the hydrolysis probe format (Hoffmann et al., 2005; Liu et al., 2007; Jamnikar Ciglenecki et al., 2008; Cheng et al., 2008) have been successfully used for an improved detection of CSFV. However, the possibility of false-negative test results poses a substantial problem in diag- nostic TaqMan assays because a single point mutation within the probe-binding site could prevent annealing of the probe and the subsequent detection (Hughes et al., 2004; Pham et al., 2005; King 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.03.022