Novel gel-based and real-time PCR assays for the improved detection of African horse sickness virus.
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 1.2TCID(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 2-3h. 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.
Rodriguez-Sanchez
Belen
B
Animal Health Department, Veterinary School, Universidad Complutense, Av. Puerta de Hierro s/n, 28040 Madrid, Spain.
Fernandez-Pinero
Jovita
J
Sailleau
Corinne
C
Zientara
Stephan
S
Belak
Sandor
S
Arias
Marisa
M
Sanchez-Vizcaino
Jose Manuel
JM
D000355:African Horse Sickness / Q000175:diagnosis / Q000821:virology; D000356:African Horse Sickness Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D052160:Benzothiazoles; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D006736:Horses; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping; D019914:Taq Polymerase; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; C089209:NS1 protein, African horse sickness virus; D009930:Organic Chemicals; D011804:Quinolines; D017361:Viral Nonstructural Proteins; C098022:SYBR Green I; D019914:Taq Polymerase