Evaluation of a multiplex real time reverse transcription PCR assay for the detection and quantitation of the most common human rotavirus genotypes

► We describe a novel real-time PCR assay for rotaviral genotyping and quantitation. ► We compare the assay with conventional semi-nested PCR. ► Two assays perform equally well. ► We provide data on rotavirus A genotype distribution and load in Greece. Group A rotaviruses (RVs) are important pathogens that cause acute, dehydrating gastroenteritis in infants and young children. In this study, a multiplex real-time polymerase chain reaction protocol using primers and TaqMan® probes specific for viral VP4 and VP7 genes was evaluated. This assay offers simultaneous genotyping and quantification of the most common RV genotypes G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]. It was compared to the molecular typing results provided by conventional PCR. A total of 92 archived stool specimens obtained from children younger than 5 years old with the diagnosis of acute gastroenteritis were examined. Real-time PCR assay detected rotavirus strains among the most common genotype combinations G4P[8] (70.7%), G1P[8] (10.9%), G2P[4] (5.4%), G9P[8] (2.2%). This new assay described has an acceptable sensitivity (low limit 6.3×102copies/g of stool).