Ljungan virus (LV), a new member of the Picornaviridae, recently isolated from vole species in both Sweden and the USA, is suspected to be pathogenic for humans as an aetiological agent in myocarditis, diabetes, neurological disease and perinatal disease. This study describes for the first time an RT-PCR assay that can identify and quantify LV infection in various tissue sample types using primers and two different minor-groove-binder probes targeting the 5'-untranslated region of the LV genome. The assay, evaluated using control samples derived from various virus cultures and rodent tissues, allows precise quantification of viral load over six orders of magnitude (10(1) to 10(6) viral copies per assay) for all known strains of LV with high sensitivity and specificity. Furthermore, a melting curve analysis (MCA) was developed using two amplicon-specific hybridisation probes that allows rapid genotyping of different LV strains. These new methods provide useful tools to investigate the putative role of LV as a pathogen in both rodents and humans.