Application of protein misfolding cyclic amplification to detection of prions in anaerobic digestate

• PMCA is an ultrasensitive tool for PrPSc detection in anaerobic digestate.
• PMCA inhibition associated with anaerobic digestate was overcome by dilution.
• PrPSc in anaerobic digestate was bioavailable for template directed amplification.
• Extraction of PrPSc was not necessary for application of PMCA or for Western blot detection.
• PMCA is applicable to complex biological matrices for evaluation of prion fate.

The exceptional physio-chemical resistance of prions to established decontamination procedures poses a challenge to assessing the suitability of applied inactivation methods. Prion detection is limited by the sensitivity level of Western blotting or by the cost and time factors of bioassays. In addition, prion detection assays can be limited by either the unique or complex nature of matrices associated with environmental samples. To investigate anaerobic digestion (AD) as a practical and economical approach for potential conversion of specified risk materials (SRM) into value added products (i.e., renewable energy), challenges associated with detection of prions in a complex matrix need to be overcome to determine potential inactivation. Protein misfolding cyclic amplification (PMCA) assay, with subsequent Western blot visualization, was used to detect prions within the AD matrix. Anaerobic digestate initially inhibited the PMCA reaction and/or Western blot detection. However, at concentrations of ≤ 1% of anaerobic digestate, 263 K scrapie prions could be amplified and semi-quantitatively detected. Infectious 263 K prions were also proven to be bioavailable in the presence of high concentrations of digestate (10–90%). Development of the PMCA application to digestate provides extremely valuable insight into the potential degradation and/or fate of prions in complex biological matrices without requiring expensive and time-consuming bioassays.