Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples

• Published in: Journal of virological methods Vol. 30; no. 1; pp. 15 - 24
• Main Authors: Wade-Evans, A.M., Mertens, P.P.C., Bostock, C.J.
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.10.1990; Amsterdam Elsevier; New York, NY
• Subjects: Animals; Base Sequence; Biological and medical sciences; blood; Bluetongue virus; Bluetongue virus - genetics; Bluetongue virus - isolation & purification; Cattle; Cell Line; Diagnosis; diagnostic techniques; DNA, Viral - analysis; DNA, Viral - genetics; Fundamental and applied biological sciences. Psychology; Microbiology; Molecular Sequence Data; Oligonucleotide Probes; PCR; polymerase chain reaction; Polymerase Chain Reaction - methods; RNA; RNA, Double-Stranded - genetics; RNA, Double-Stranded - isolation & purification; RNA, Viral - genetics; RNA, Viral - isolation & purification; Techniques used in virology; Virology; PCR; Diagnosis; Bluetongue virus; Infection; Virus; Molecular hybridization; Tissue; Complementary DNA; RNA; Viral disease; Orbivirus; Detection; Reoviridae; Optimization
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/0166-0934(90)90040-M

Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTV1SA), was used as template to optimise a polymerase chain reaction (PCR) for the detection of bluetongue virus RNA. Pairs of oligonucleotides complementary to the 3' termini of eight of the ten genome segments were tested. Those representing the 5' termini of genome segment 7 gave the best amplification results producing a single DNA band with the same mobility during agarose gel electrophoresis as genome segment 7. It was confirmed by cloning and sequence analysis, that this PCR-amplified DNA contained both terminal regions of genome segment 7 and therefore represented full length cDNA. Using these segment 7 oligonucleotides it was not only possible to detect routinely as few as 6 molecules of segment 7 dsRNA per sample, but also to detect purified dsRNAs from isolates of other BTV serotypes (1 Australia (AUS), 2, 3, 4, 10, 16 and 20). However, with the exception of Tilligery virus, isolates from other Orbivirus serogroups tested all gave negative results (African horse sickness, epizootic haemorrhagic disease, Palyam, Warrego and Eubenangee). The PCR was also used to analyse red blood cells (RBC) and buffy coat samples from cattle infected with BTV4. Positive results were obtained from samples taken 7 days post-infection (p.i.) (containing 1.6 × 10 3 TCID 50 of virus/ml of whole blood) and from the RBC sample only, taken 14 days p.i. (16 TCID 50/ml). However, at 28 days p.i. (< 1.6 TCID 50/ml) BTV RNA was not detected using the PCR in either sample.