Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae

S M Da Silva; L K Vang; N D Olson; S P Lund; A S Downey; Z Kelman; M L Salit; N J Lin; J B Morrow

doi:10.1016/j.bdq.2016.01.001

Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae

Biomol Detect Quantif. 2016 Feb 19:7:27-33. doi: 10.1016/j.bdq.2016.01.001. eCollection 2016 Mar.

Authors

S M Da Silva¹, L K Vang¹, N D Olson¹, S P Lund², A S Downey¹, Z Kelman³, M L Salit⁴, N J Lin¹, J B Morrow¹

Affiliations

¹ Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
² Statistical Engineering Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
³ Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, USA; Institute for Bioscience and Biotechnology Research, Rockville, MD, USA.
⁴ Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA; Department of Bioengineering, Stanford University, Stanford, CA, USA.

Abstract

Aims: We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR).

Methods and results: S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 10(7) cells ml(-1)), intermediate (4 × 10(5) cells ml(-1)) and low (4 × 10(3) cells ml(-1)), and the number of samples per concentration. Seven participants, including potential end users, had combined rates of positive qPCR detection (quantification cycle <37) of 100%, 40%, and 0% for high, intermediate, and low concentrations, respectively.

Conclusions: The NE095 strain was successfully detected by all participants, with the high concentration indicating a potential target concentration for a reference material.

Significance and impact of the study: The engineered yeast has potential to support measurement assurance for the analytical process of qPCR, encompassing the method, equipment, and operator, to increase confidence in results and better inform decision-making in areas of applied microbiology. This material can also support process assessment for other DNA-based detection technologies.

Keywords: ERCC; Evaluation material; Interlaboratory study; Reference material; Saccharomyces cerevisiae; qPCR.