Novel gel-based and real-time PCR assays for the improved detection of African horse sickness virus

• Published in: Journal of virological methods 2008-07, Vol.151 (1), p.87-94
• Main Authors: Rodriguez-Sanchez, Belen, Fernandez-Pinero, Jovita, Sailleau, Corinne, Zientara, Stephan, Belak, Sandor, Arias, Marisa, Sanchez-Vizcaino, Jose Manuel
• Format: Article
• Language: English
• Subjects: African horse sickness; African Horse Sickness - diagnosis; African Horse Sickness - virology; African horse sickness virus; African Horse Sickness Virus - classification; African Horse Sickness Virus - genetics; African Horse Sickness Virus - isolation & purification; Animals; Biological and medical sciences; Bluetongue virus; Cercopithecus aethiops; DNA Primers; DNA, Viral - analysis; Electrophoresis, Agar Gel; Fundamental and applied biological sciences. Psychology; Horses; Life Sciences; Microbiology; Microbiology and Parasitology; Non-structural protein NS1; Organic Chemicals; Polymerase Chain Reaction - methods; Real-time RT-PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity; Serotype; Serotyping; SYBR-Green; Taq Polymerase; TaqMan; Techniques used in virology; Vero Cells; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - genetics; Virology; West Nile virus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2008.03.029

In order to improve, ensure and accelerate the diagnosis of African horse sickness, a highly devastating, transboundary animal disease listed by the World Animal Health Organisation, (OIE) three novel diagnostic PCR assays were developed and tested in this study. The reverse transcription-PCR (RT-PCR) tests were the following: (a) a conventional, gel-based RT-PCR, (b) a real-time PCR with SYBR-Green-named rRT-PCR SYBR-Green-, and (c) a real-time PCR rRT-PCR with TaqMan ® probe (termed rRT-PCR TaqMan ®). The same pair of primers—directed against African Horse Sickness Virus (AHSV) segment 5, encoding the non-structural protein NS1, is used in the three tests listed above. The three PCR assays detected similarly the nine AHSV serotypes from cultivated viral suspensions of different origins. The RT-PCR assays provided high sensitivity ranging from 0.1 to 1.2 TCID 50/ml. The specificity was also high, considering that related viruses, such as Bluetongue virus, and other equine viruses, such as West Nile Virus, remained negative for RT-PCR amplification. The detection of AHSV virus can be completed within 2–3 h. These results indicate that the novel PCR methods described in this paper provide robust and versatile tools that allow rapid and highly specific, simultaneous detection of all AHSV serotypes.