Rapid genomic typing of BK virus directly from clinical specimens

Abstract

A simple and rapid method, PCR-restriction enzyme analysis (PCR-RE) for BK virus (BKV) typing was developed, based on the presence of type-specific restriction enzyme sites in a 327 bp PCR-generated fragment which partially encodes the VP1 protein. The enzymes, Alu I, Xmn I and Ava II, were used to digest the PCR products in two stages. Ethidium bromide banding patterns characteristic of each of four subtypes of BKV were visualized through gel electrophoresis. A total of 37 samples from clinical specimens and culture fluids were successfully subtyped using the PCR-RE. A second method, PCR-sequencing, was applied to samples that generated less than 100 ng of DNA. These were subjected to a second round of PCR and then sequenced from single stranded templates immobilised via a biotinylated primer. The subtypes were assigned on the basis of the type-specific sequences previously characterized.