Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve
Version of Record online: 28 JUN 2011
© 2011 The Societies and Blackwell Publishing Asia Pty Ltd
Microbiology and Immunology
Volume 55, Issue 7, pages 502–509, July 2011
How to Cite
Takeuchi, A., Komiya, M., Kitamoto, T. and Morita, M. (2011), Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve. Microbiology and Immunology, 55: 502–509. doi: 10.1111/j.1348-0421.2011.00340.x
- Issue online: 28 JUN 2011
- Version of Record online: 28 JUN 2011
- Accepted manuscript online: 28 MAR 2011 11:44AM EST
- Received 15 December 2010; revised 8 February 2011; accepted 8 March 2011.
- protein misfolding cyclic amplification
In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8–7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.