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

• Published in: Journal of virological methods Vol. 174; no. 1-2; pp. 53 - 59
• Main Authors: Pérez, Lester Josué, Díaz de Arce, Heidy, Tarradas, Joan, Rosell, Rosa, Perera, Carmen Laura, Muñoz, Marta, Frías, Maria T., Nuñez, José Ignacio, Ganges, Llilianne
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.06.2011; Elsevier
• Subjects: Animals; Biological and medical sciences; blood serum; cell culture; Classical Swine Fever - diagnosis; Classical Swine Fever - virology; Classical swine fever virus; Classical swine fever virus - genetics; Classical swine fever virus - isolation & purification; Clinical Laboratory Techniques - methods; culture media; detection limit; disease control; financial economics; Fundamental and applied biological sciences. Psychology; genes; genotype; hog cholera; melting; Microbiology; Organic Chemicals - metabolism; quantitative polymerase chain reaction; Real-time PCR instrument; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - genetics; RNA, Viral - isolation & purification; Sensitivity and Specificity; Staining and Labeling - methods; Swine; swine production; SYBR Green real-time RT-PCR; Techniques used in virology; Virology; Virology - methods; viruses; SYBR Green real-time RT-PCR; Real-time PCR instrument; Classical swine fever virus; Pestivirus; Method; Real time; Virus; Polymerase chain reaction; Flaviviridae; Detection; Veterinary; Reverse transcription polymerase chain reaction; Hog cholera virus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2011.03.022

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 analytical 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 102 gene copies/μL 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.