Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.

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.

SUBMITTER: Da Silva SM 

PROVIDER: S-EPMC4827694 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature