Sensitive and specific nested PCR assay for detection of rotavirus A in samples with a low viral load

Techniques such as the real-time reverse transcription-polymerase chain reaction (qRT-PCR) can detect RNA in samples with a low viral load. However, these amplicons typically are either too short or at insufficient concentrations for use in subsequent sequencing reactions for genotyping and detection confirmation. The assay developed in this study detects rotavirus G genotypes and P genotypes with viral loads as low as 6.2 and 8.2 copies per reaction, respectively. The assay was validated using a panel of 91 stool samples, 32 reference rotavirus strains, and 6 non-target enteric virus samples.