SummaryPrion diseases are fatal neurodegenerative diseases that affect mammals through the transconformation of a host protein, the prion protein (PrP), into a toxic and pathogenic conformer termed PrPSc. Until now, the diagnosis is only confirmed with a post-mortem histology study of the central nervous system. Among the methods to detect the etiological agent,in vitroamplification techniques have emerged as very sensitive, highly specific and rapid tools, even though some prion strains remain refractory or difficult to amplify. Here we report the use of a new recombinant substrate for Real-Time Quaking Induced Conversion (RT-QuIC), a natural polymorphism of human prion protein with a lysine at position 219 instead of a glutamic acid, PrP E219K. This substrate amplifies the six sporadic human strains responsible for Creutzfeldt-Jakob Disease (CJD) and the strain responsible for its variant form in a few hours and over a large dilution range of the seeds. Moreover, based on the lag time of the amplification reactions, the PrP E219K substrate allows to discriminate between sporadic and variant CJD strains, a first step towards an ante-mortem typing of the prion strain affecting a patient.