Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of foot-and-mouth disease virus.
Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting the 3D gene of FMDV. The assay was validated for the efficacy to detect all known FMDV serotypes. The test method was linear over a range of at least 7 orders of magnitude and the detection limit was below the equivalent of 10 genomic copies. Analysing recent African probang samples the method was able to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon by melting point analysis for added specificity and at the same time allows the detection of mutations in the probe region. As such, the described new method is suitable for the robust real-time detection of index cases caused by any serotype of FMDV.
Rasmussen
T B
TB
Department of Virology, Danish Veterinary Institute, Kalvehave, Denmark.
Uttenthal
A
A
de Stricker
K
K
Belák
S
S
Storgaard
T
T
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D002020:Buffaloes; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004735:Energy Transfer; D005456:Fluorescent Dyes; D005536:Foot-and-Mouth Disease / Q000175:diagnosis* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012703:Serotyping; D017361:Viral Nonstructural Proteins / Q000235:genetics
D000956:Antigens, Viral; D017931:DNA Primers; D005456:Fluorescent Dyes; D017361:Viral Nonstructural Proteins; C103557:virus-infection-associated antigen, Foot-and-mouth disease virus