ultra-efficient prpsc amplification highlights potentialities and pitfalls of pmca technology高效prpsc放大突出pmca技术的潜力和陷阱.pdfVIP

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ultra-efficient prpsc amplification highlights potentialities and pitfalls of pmca technology高效prpsc放大突出pmca技术的潜力和陷阱.pdf

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ultra-efficient prpsc amplification highlights potentialities and pitfalls of pmca technology高效prpsc放大突出pmca技术的潜力和陷阱

Ultra-Efficient PrPSc Amplification Highlights Potentialities and Pitfalls of PMCA Technology 1,2 1 1 1 Gian Mario Cosseddu *, Romolo Nonno , Gabriele Vaccari , Cecilia Bucalossi , Natalia Fernandez- 2,3 1 2,3,4 1 Borges , Michele Angelo Di Bari , Joaquin Castilla , Umberto Agrimi ` 1 Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanita, Rome, Italy, 2 Department of Infectology, The SCRIPPS Research Institute, Jupiter, ´ Florida, United States of America, 3 33458CIC bioGUNE, Parque Tecnologico de Bizkaia, Derio, Spain, 4 IKERBASQUE, Basque Foundation for Science, Bilbao, Spain Abstract In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK- resistant PrPSc in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions.