New oligonucleotide primers for P-typing of rotavirus strains: Strategies for typing previously untypeable strains

Background: The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing, and allows the identification of putative reassortant strains. However, due to the constant accumulation of point mutations through genetic drift; and to the emergence of novel genotypes; and possibly zoonotic transmission and subsequent reassortment, the reagents and methods used for genotyping require close monitoring and updating. Objectives: To design and evaluate a new VP4 consensus oligonucleotide primer pair that provides increased sensitivity and allows typing of strains that were untypeable using available methods. Study design: A total of 489 rotavirus-positive faecal specimens from studies conducted between 1996 and 2006 were used for the evaluation of the new VP4 primers which was performed in the WHO Rotavirus Collaborating and Reference centres in the US, Australia, South Africa and the UK. Results: The new primer pair allowed P-typing of rotavirus strains and provided increased sensitivity, allowing typing of a significant number of strains that previously could not be P-typed. Conclusions: This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.