Development of a novel PCR-RFLP assay for improved detection and typing of bovine papillomaviruses.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and type bovine papillomaviruses (BPVs) from tumors in cattle. Two degenerate primer sets targeting the BPV L1 gene, subAup/subAdw and subBup/subBdw, and one restriction enzyme RsaI were used in this assay. In silico analyses of the restriction enzyme sites in the PCR fragments of 13 BPV sequences (BPV-1 to -13) revealed that all known BPVs are differentiated by the PCR-RFLP assay. Analyses of 63 previously typed clinical samples, that included teat papillomas and both esophageal and urinary bladder cancer biopsies, show that the assay clearly differentiates between eight clinically important BPV types (BPV-1 to -6, -9, -10), and discriminates between single and multiple infections. To further assess the reliability of the PCR-RFLP method amplified fragments were sequenced. A high correlation (95%) was observed when the results of the PCR-RFLP method were compared with PCR-sequencing. Differences in typing occurred for 3 of 63 specimens; PCR-RFLP identified additional BPV types in these specimens, while the PCR-sequencing identified only one. These results indicate that the PCR-RFLP method reported here is simpler and more reliable in the detection and typing of BPVs from bovine tumor samples than PCR-sequencing.