Detection of bluetongue virus serogroup by polymerase chain reaction.
To facilitate detection of active bluetongue virus (BTV) infection, a polymerase chain reaction (PCR) protocol was developed. The BTV reverse transcriptase PCR (RT-PCR) is a 1-tube reaction and involves chemical denaturation of the double-stranded viral RNA target, a complementary DNA (cDNA) synthesis step, and PCR amplification of the cDNA. BTV RT-PCR using primers derived from highly conserved genome segment 10 results in a 251-base pair (bp) product. BTV RNA from all USA prototype serotypes 2, 10, 11, 13, and 17; a wide spectrum of USA BTV field isolates including serotypes 10, 11, 13, and 17; and a spectrum of Israeli field isolates including serotypes 2, 4, 6, 10, and 16 were detected by BTV RT-PCR. With agarose gels, the 251-bp product was detected from as little as 100 fg-1 pg of BTV RNA, which is equivalent to 5 x 10(3)-5 x 10(4) viral particles or 5 x 10(2)-5 x 10(3) infectious units. With dot blot hybridization, specific PCR product was detected from as little as 1 fg of BTV RNA, which is equivalent to 50 viral particles, or 5 infectious units. This level of sensitivity is comparable to that of virus isolation. The BTV RT-PCR using primers derived from genome segment 10 can detect a wide spectrum of USA and Israeli BTV serotypes and has potential for detection of infection by the BTV serogroup. Application of this BTV PCR to clinical samples is in progress.
D001483:Base Sequence; D001820:Bluetongue virus / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA; D012329:RNA, Bacterial / Q000235:genetics*; D012680:Sensitivity and Specificity
C034477:RNA primers; D012329:RNA, Bacterial; D012313:RNA