title: Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.
abstract: OBJECTIVE
  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).
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Da Silva
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: L K
authors.initials: LK
authors.lastname: Vang
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: N D
authors.initials: ND
authors.lastname: Olson
authors.affiliation: Statistical Engineering Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Lund
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Downey
authors.affiliation: Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, USA; Institute for Bioscience and Biotechnology Research, Rockville, MD, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Kelman
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA; Department of Bioengineering, Stanford University, Stanford, CA, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Salit
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Lin
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Morrow
keywords: ERCC; Evaluation material; Interlaboratory study; Reference material; Saccharomyces cerevisiae; qPCR