Detection and quantitation of bluetongue virus serotypes by a TaqMan probe-based real-time RT-PCR and differentiation from epizootic hemorrhagic disease virus.
Twenty-four serotypes of bluetongue virus (BTV) have been recognized world wide. Reliable and quantitative assays of virus universal detection are essential for fighting against BT. A real-time reverse transcription-polymerase chain reaction (RT-PCR) with a TaqMan fluorescence probe has been developed for detection of the NS1 gene of different BTV serotypes. In BHK-21 cells, in the assay detected BTV1-22 specifically, and had no cross-reactivity with the closely related epizootic hemorrhagic disease virus (EHDV) serotypes 1-5. The limit of sensitivity of the assay was 0.1 TCID(50)/ml for BTV-1 and 10(2) copies for the control R121/pGEM. Accurate quantitation can be achieved with samples containing between 10(2) and 10(6) copies. The coefficient of variation (CV) of intra-assay and inter-assay ranged from 2.17% to 5.60%. The developed real-time RT-PCR assay showed good coincident rate (99.2%) with duplex RT-PCR in 122 whole blood clinical samples from sheep. Therefore, the real-time RT-PCR can be a reliable method for detection of various serotypes of BTV.
Yin
Hui-qiong
HQ
Viral Safety Laboratory of National Center of Biomedical Analysis, Institute of Transfusion Medicine, the Academy of Military Medical Sciences, Beijing 100850, China.
Zhang
Hong
H
Shi
Li-jun
LJ
Yang
Shu
S
Zhang
Gai-ping
GP
Wang
Sheng-qi
SQ
Zhang
Jin-gang
JG
D000818:Animals; D001819:Bluetongue / Q000175:diagnosis*; D001820:Bluetongue virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005453:Fluorescence; D018164:Hemorrhagic Disease Virus, Epizootic / Q000235:genetics / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
C078966:NS1 protein, bluetongue virus; D015345:Oligonucleotide Probes; D017361:Viral Nonstructural Proteins