For parechovirus molecular typing, we sought to develop reverse transcription, nested polymerase chain reaction (RT-PCR) assays to amplify the sequence encoding the VP1 capsid protein from all known members of the Parechovirus genus.
The assays consist of a two-step RT-PCR with primers flanking VP1 (PCR1), followed by semi-nested PCR2A and PCR2B reactions that produce overlapping amplicons, encompassing the complete VP1 gene, as well as a nested PCR2C that amplifies a shorter internal VP1 amplicon.
All primer sets are 100 % sensitive and 100 % specific for the 77 parechovirus culture isolates tested. The semi-nested and nested PCR primer sets are 94 % sensitive and 100 % specific for detection of parechovirus in original specimens. Viral genotype can be deduced from analysis of amplicon sequences. Parechoviruses of the same type share ≥77 % complete VP1 nucleotide sequence identity or ≥87 % amino acid identity, while those of different types share ≤73 % nucleotide identity and ≤81 % amino acid identity.
The PCR primers described here amplify VP1 sequences from all known parechoviruses, providing a sensitive, reliable system for molecular typing directly from original clinical specimens.