Nested reverse transcriptase-polymerase chain reaction for the detection of group A rotaviruses.
Rotaviruses are important pathogens associated with diarrhoeal diseases in almost all species of mammals. In the present study, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of group A rotaviruses was developed. It is based on a target region in gene segment 6. Rotavirus strains of human, bovine, porcine, canine, feline, equine and ovine origin were examined. Furthermore, several faecal specimens, in which rotavirus had already been detected using other methods than PCR, were included in the study. A nested RT-PCR product was formed with all strains and faecal samples tested. The detection limit for virus-containing cell culture supernatant was 3×10-2 (50% tissue culture infective dose, TCID50) by RT-PCR and 3×10-3 TCID50 by nested amplification. In order to examine the influence of the sample matrix on sensitivity, a rotavirus-negative faecal specimen was spiked with virus-containing cell culture suspension of the porcine rotavirus OSU. The detection limit of the present PCR procedure was approximately 1.6×102 TCID50 per gram faeces, and could be increased by one order of magnitude using nested PCR. The present method for the detection and identification of group A rotaviruses represents a powerful diagnostic tool and was shown to be applicable to rotaviruses of different origin, including human sources.