A single-tube RT-PCR for rapid detection and differentiation of some African isolates of palyam serogroup orbiviruses

A single-tube nested reverse transcriptase (nRT) polymerase chain reaction (nRT-PCR) was developed and evaluated for detection of palyam serogroup orbiviruses ribonucleic acid (RNA) in cell cultures and clinical samples. A pair of outer primers (pal1 and pal2), designed from genome segment three of Chuzan virus of the palyam viruses serogroup, resulted in amplification of a primary 660-base pair (bp) PCR product. Using a pair of internal (nested) primers (pal3 and pal4), the nRT-PCR produced a 350-bp PCR product. The primary and the nested PCR products were amplified from RNA extracted from Sudanese and South African isolates of palyam viruses, propagated in cell cultures. Application of this nRT-PCR to clinical samples resulted in direct detection of palyam virus RNA in blood and serum samples from infected cattle and goats. The nested amplification increased the sensitivity of the assay by 1000-fold, and specific PCR products were detected from as little as 0.1 fg of viral RNA. Amplification products were not detected when the nRT-PCR was applied to RNA from closely related orbiviruses including, bluetongue virus (BTV) serotypes 1, 2, 4, 6; epizootic hemorrhagic disease of deer virus prototype serotype 1 (EHDV-1); Sudanese isolates of EHDV-318; total nucleic acid extracts from non-infected Vero cells; and blood and sera from goats and calves from which virus was not isolated. This nRT-PCR provides a reliable, sensitive and specific assay for rapid detection and differentiation of palyam viruses from other related orbiviruses. In addition, the assay is recommended for inclusion in epidemiological surveys and during investigation of an epizootic of the disease among susceptible ruminants.