Evaluation of a reverse line blot assay for genotyping common human rotaviruses

To allow high-throughput genotyping of group A rotaviruses (RVA) in a routine surveillance setting, we developed a reverse line blotting method for the determination of the most common human RVA G- and P-genotypes: G1-G4, G9, G12, P[4], P[6] and P[8]. Using the reverse line blotting method on 951 clinical RVA positive feces samples, in 905 (95%) of the samples the G-genotyping yielded a result while in 945 (99%) of the samples the P-genotyping was successful. Comparison of the reverse line blotting-method as it is used currently to a sequence based method for genotyping RVAs showed an agreement of 96% for single strain infections (75 out of 78) but only 48% for mixed infections (10 out of 21).The reverse line blotting method is successful in genotyping common RVA strains in surveillance settings. For genotyping of rare strains, the number of probes on the blot can be expanded or a sequence-based method can be performed as a complementary approach on the samples that are positive in the detection PCR but negative in the reverse line blotting genotyping assay. The complete reverse line blotting protocol takes 8. h to complete for 36 samples, with 3. h hands-on-time. In conclusion, a RVA genotyping method that is accurate, cheap and fast is presented.

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