Rotavirus is a dsRNA virus that causes gastroenteritis. Clinical fecal samples, previously confirmed to contain rotavirus, were used to test the design strategies of this report. An asymmetric PCR, with biotinylated rev-primer in excess, was set up for the VP6 segment. The primers were designed using knowledge gained from this report, i.e. both degeneration and dInosine were used to create moderately degenerated probes with uninterrupted matching stretches as long as possible, in the most conserved regions. The biotinylated reverse (rev-) primer was made 77-nt long to be as tolerant to variation as possible, while the shorter forward (fw-) primer (23 nt) contained four locked nucleic acids (LNA) to increase the hybridization strength (represented by melting temperature, T m ) to match that of the rev-primer.