High throughput detection of bluetongue virus by a new real-time fluorogenic reverse transcription-polymerase chain reaction: application on clinical samples from current Mediterranean outbreaks.
A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of bluetongue virus (BTV) in blood samples. A combination of primers specific for a highly conserved region in RNA segment 5 (based on Mediterranean BTV sequences) and a DNA probe bound to 5'-Taq nuclease-3' minor groove binder (TaqMan MGB) was used to detect a range of isolates. This real-time RT-PCR assay could detect 5.4 x 10(-3) tissue culture infectious doses (TCID50) of virus per milliliter of sample, which was comparable to our current BTV diagnostic nested RT-PCR assay. The assay detected all recent Mediterranean isolates (including serotypes 2, 4, and 16), BTV vaccine strains for serotypes 2 and 4, and 15 out of the 24 BTV reference strains available (all serotypes), but did not detect the related orbiviruses epizootic hemorrhagic disease and African horse sickness viruses. Following assay evaluation, the ability of this assay to identify BTV in recent isolates (2003, 2004) from ovine and bovine samples from an epizootic outbreak in Spain was also tested. Minor nucleotide changes (detected by sequencing viral genomes) within the probe-binding region were found to have a profound effect on virus detection. This assay has the benefits of being fast and simple, and the 96-well format enables large-scale epidemiological screening for BTV, especially when combined with a high-throughput nucleic acid extraction method.
Departamento de Enfermedades Emergentes, Laboratorio Central de Veterinaria, Algete, Madrid, Spain.
Miguel Angel
MA
Jiménez-Clavero
Montserrat
M
Agüero
Elena
E
San Miguel
Tomás
T
Mayoral
Maria Cruz
MC
López
María José
MJ
Ruano
Esther
E
Romero
Federica
F
Monaco
Andrea
A
Polci
Giovanni
G
Savini
Concepción
C
Gómez-Tejedor
D000818:Animals; D001483:Base Sequence; D001819:Bluetongue / Q000175:diagnosis* / Q000453:epidemiology; D001820:Bluetongue virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D004196:Disease Outbreaks / Q000662:veterinary; D019083:Mediterranean Region / Q000453:epidemiology; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification*; D012044:Regression Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012418:Ruminants; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D012703:Serotyping / Q000662:veterinary; D012756:Sheep; D013030:Spain / Q000453:epidemiology
D015342:DNA Probes; D012367:RNA, Viral