Rapid and sensitive detection of African horse sickness virus by real-time PCR.
A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID(50) per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of field equine samples. The same primer pair was used to standardise a conventional RT-PCR as an affordable, useful and simple alternative method in laboratories without access to real-time PCR instruments. The two techniques present novel tools to improve the molecular diagnosis of African horse sickness (AHS).
Fernández-Pinero
J
J
Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain. fpinero@inia.es
Fernández-Pacheco
P
P
Rodríguez
B
B
Sotelo
E
E
Robles
A
A
Arias
M
M
Sánchez-Vizcaíno
J M
JM
D000355:African Horse Sickness / Q000235:genetics / Q000821:virology*; D000356:African Horse Sickness Virus / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D006736:Horses; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013030:Spain
D012367:RNA, Viral