Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.

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.

Single-element solution comparisons with a high-performance inductively coupled plasma optical emission spectrometric method.

A solution-based inductively coupled plasma optical emission spectrometric (ICP-OES) method is described for elemental analysis with relative expanded uncertainties on the order of 0.1% relative. The single-element determinations of 64 different elements are presented, with aggregate performance results for the method and parameters for the determination of each element. The performance observed is superior to that previously reported for ICP-OES, resulting from a suite of technical strategies that exploit the strengths of contemporary spectrometers, address measurement and sample handling noise sources, and permit rugged operation with small uncertainty. Taken together, these strategies constitute high-performance ICP-OES.

Using inductively coupled plasma-mass spectrometry for calibration transfer between environmental CRMs.

Multielement analyses of environmental reference materials have been performed using existing certified reference materials (CRMs) as calibration standards for inductively coupled plasma-mass spectrometry. The analyses have been performed using a high-performance methodology that results in comparison measurement uncertainties that are significantly less than the uncertainties of the certified values of the calibration CRM. Consequently, the determined values have uncertainties that are very nearly equivalent to the uncertainties of the calibration CRM. Several uses of this calibration transfer are proposed, including, re-certification measurements of replacement CRMs, establishing traceability of one CRM to another, and demonstrating the equivalence of two CRMs. RM 8704, a river sediment, was analyzed using SRM 2704, Buffalo River Sediment, as the calibration standard. SRM 1632c, Trace Elements in Bituminous Coal, which is a replacement for SRM 1632b, was analyzed using SRM 1632b as the standard. SRM 1635, Trace Elements in Subbituminous Coal, was also analyzed using SRM 1632b as the standard.

A drift correction procedure.

A procedure is introduced that can mitigate the deleterious effect of low-frequency noise [Formula: see text] often termed drift [Formula: see text] on the precision of an analytical experiment. This procedure offers several performance benefits over traditional designs based on the periodic measurement of standards to diagnose and correct for variation in instrument response. Using repeated measurements of every sample as a drift diagnostic, as opposed to requiring the periodic measurement of any given sample or standard, the analyst can better budget the measurement time to be devoted to each sample, distributing it to optimize the uncertainty of the analytical result. The drift is diagnosed from the repeated measurements, a model of the instrument response drift is constructed, and the data are corrected to a "drift-free" condition. This drift-free condition allows data to be accumulated over long periods of time with little or no loss in precision due to drift. More than 10-fold precision enhancements of analytical atomic emission results have been observed, with no statistically significant effects on the means. The procedure is described, performance data are presented, and matters regarding the procedure are discussed.

Wavelengths of spectral lines in mercury pencil lamps.

The wavelengths of 19 spectral lines in the region 253-579 nm emitted by Hg pencil-type lamps were measured by Fourier-transform spectroscopy. Precise calibration of the spectra was obtained with wavelengths of (198)Hg as external standards. Our recommended values should be useful aswavelength-calibration standards for moderate-resolution spectrometers at an uncertainty level of 0.0001 nm.

Practical wavelength calibration considerations for UV-visible Fourier-transform spectroscopy.

The intrinsic wavelength scale in a modern reference laser-controlled Michelson interferometer-sometimes referred to as the Connes advantage-offers excellent wavelength accuracy with relative ease. Truly superb wavelength accuracy, with total relative uncertainty in line position of the order of several parts in 10(8), should be within reach with single-point, multiplicative calibration. The need for correction of the wavelength scale arises from two practical effects: the use of a finite aperture, from which off-axis rays propagate through the interferometer, and imperfect geometric alignment of the sample beam with the reference beam and the optical axis of the moving mirror. Although an analytical correction can be made for the finite-aperture effect, calibration with a trusted wavelength standard is typically used to accomplish both corrections. Practical aspects of accurate calibration of an interferometer in the UV-visible region are discussed. Critical issues regarding accurate use of a standard external to the sample source and the evaluation and selection of an appropriate standard are addressed. Anomalous results for two different potential wavelength standards measured by Fabry-Perot interferometry (Ar II and (198)Hg I) are observed.