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Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae
S M Da Silva1, L K Vang1, N D Olson1
1Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
Biomolecular Detection and Quantification
|April 15, 2016
Summary
An engineered yeast strain, Saccharomyces cerevisiae NE095, was developed for quantitative polymerase chain reaction (qPCR) assays. This material shows promise for validating DNA detection workflows and improving measurement assurance in applied microbiology.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Quantitative polymerase chain reaction (qPCR) is a vital tool in applied microbiology.
- Standardized materials are needed to ensure the reliability of complex DNA detection workflows.
Purpose of the Study:
- To develop and evaluate a modified Saccharomyces cerevisiae strain (NE095) as a candidate reference material.
- To assess its utility in an interlaboratory study for validating DNA extraction and qPCR processes.
Main Methods:
- Engineered S. cerevisiae NE095 by inserting a specific DNA sequence into S. cerevisiae BY4739.
- Conducted an interlaboratory study with seven participants using three defined cell concentrations (high, intermediate, low).
- Analyzed qPCR detection rates using a binomial regression model.
Main Results:
- The NE095 strain was consistently detected at high concentrations (100% detection rate).
- Detection rates decreased significantly at intermediate (40%) and low (0%) concentrations.
- The high concentration proved effective for target detection in the interlaboratory study.
Conclusions:
- The engineered NE095 yeast strain is a viable candidate for a reference material to support qPCR measurement assurance.
- This material can enhance confidence in results across the entire analytical process (method, equipment, operator).
- It also aids in process assessment for other DNA-based detection technologies.
