Development of Seven New dPCR Animal Species Assays and a Reference Material to Support Quantitative Ratio Measurements of Food and Feed Products.

Laboratory testing methods to confirm the identity of meat products and eliminate food fraud regularly rely on PCR amplification of extracted DNA, with most published assays detecting mitochondrial sequences, providing sensitive presence/absence results. By targeting single-copy nuclear targets instead, relative quantification measurements are achievable, providing additional information on the proportions of meat species detected. In this Methods paper, new assays for horse, donkey, duck, kangaroo, camel, water buffalo and crocodile have been developed to expand the range of species that can be quantified, and a previously published reference assay targeting the myostatin gene has been modified to include marsupials and reptiles. The accuracy of this ratio measurement approach was demonstrated using dPCR with mixtures of meat DNA down to 0.1%. However, the limit of detection (LOD) of this approach is not just determined by the assay targets, but by the samples themselves, with food or feed ingredients and processing impacting the DNA yield and integrity. In routine testing settings, the myostatin assay can provide multiple quality control roles, including monitoring the yield and purity of extracted DNA, identifying the presence of additional meats not detected by the suite of species-specific assays and potentially estimating a sample-specific LOD based on measured copy numbers of the myostatin target. In addition to the myostatin positive control assay, a synthetic DNA reference material (RM) has been designed, containing PCR targets for beef, pork, sheep, chicken, goat, kangaroo, horse, water buffalo and myostatin, to be used as a positive template control. The availability of standardised measurement methods and associated RMs significantly improves the reliability, comparability and transparency of laboratory testing, leading to greater confidence in results.

Droplet Volume Variability and Impact on Digital PCR Copy Number Concentration Measurements.

Digital polymerase chain reaction (dPCR) is increasingly being adopted by reference material producers and metrology institutes for value assignment, and for homogeneity and stability studies of nucleic acid reference materials. A reference method procedure should fulfill several requirements, and the uncertainty and biases should be completely understood. A bias in target concentration when inaccurate droplet volume is used in the droplet dPCR measurement equation has previously been documented. In this study, we characterize both intrawell and interwell droplet volume variability using optical microscopy and determine the impact of these two sources of variability on target concentration estimates. A small optical distortion across the image was measured which, without correction, biased droplet volume measurements. Longitudinal monitoring of interwell droplet volume over 39 weeks using several lots of Mastermix demonstrated a mean droplet volume of 0.786 nL and intermediate precision of 1.7%. The frequency distribution of intrawell droplet volumes varied. Some wells displayed a skewed distribution which resulted in a small bias in estimated target concentration for a simulated dPCR with target concentrations of between 62 and 8000 copies µL-1. The size and direction of this bias was influenced by the distribution pattern of the droplet volumes within the well. The proportion of Mastermix in dPCR mix affected droplet volume. A pipetting error of 10% during mixing of the premix and Mastermix resulted in a 2.6% change in droplet volume and, consequently, a bias in concentration measurements highlighting the advantages of gravimetric preparation of dPCR mixes for high accuracy measurements.

Digital polymerase chain reaction measured pUC19 marker as calibrant for HPLC measurement of DNA quantity.

Digital polymerase chain reaction (dPCR) is potentially a primary method for quantifying target DNA regions in a background of nontarget material and is independent of external calibrators. Accurate dPCR measurements require single-molecule detection by conventional PCR assays that may be subject to bias due to inhibition, interference, or sequence-derived PCR inefficiency. Elimination or control of such biases is essential for validation of PCR assays, but this may require a substantial investment in resources. Here we present a mechanism for DNA quantification that does not require PCR assay validation in situations where target DNA quantity is high enough to be measured by physical techniques such as quantitative high-performance liquid chromatography (HPLC) or electrophoresis. A commercially available DNA marker derived from pUC19 was quantified by dPCR and was then used to calibrate an HPLC measuring system for quantifying a DNA amplicon that had a high content of guanidine and cytidine. The dPCR-calibrated HPLC measurement was verified by independent measurement using isotope dilution mass spectrometry (IDMS). HPLC quantification, calibrated with dPCR or IDMS measured DNA markers, provides an effective method for certifying the quantity of genetic reference materials that may be difficult to analyze by PCR. These secondary reference materials may then be used to validate and calibrate quantitative PCR measurements and thus could expand the breadth of applications for which traceability to the International System of Units is possible.

Accurate measurement of DNA methylation that is traceable to the international system of units.

The increased presence of 5-methycytosine at gene promoter regions may be diagnostic of cancer. However, there are many stages in the measurement of gene promoter 5-methylcytosine content where inaccuracies may occur, and this may prevent the use of these measurements for diagnostic or prognostic purposes. A high accuracy LC-MS system was developed for measuring the degree of methylation in two 100 base pair amplicons generated by the polymerase chain reaction (PCR) and in which 5-methylcytidine had been synthetically incorporated. Nucleotide monophosphate reference materials were used to calibrate the peak area ratio of cytidine and 5-methylcytidine to their mole ratio in enzymatic hydrolysates of the amplicons, thus enabling metrological traceability of the methylation ratio to the mole. The methylation values obtained agreed closely with the reference values assigned to the materials. A measurement uncertainty budget was completed and showed that the moisture content of the nucleotide monophosphate reference materials was the largest source of uncertainty in the methylation ratio measurement. Measurement of an oligonucleotide supplied with the materials provided evidence that such materials may be used for calibration of DNA methylation ratios without the need for measurement of moisture content. This raises the possibility that submicrogram amounts of appropriately characterized oligonucleotide reference materials could be used to calibrate methylation ratios obtained by contemporary methodologies (such as PCR after bisulfite conversion of genomic DNA) yielding values that are traceable to the International System of Units (SI). Such calibrated gene methylation measurements would then be internationally comparable as required for effective diagnostic and prognostic measurements.

Development of a measurement system for certifying ethanol mass fraction in aqueous solutions.

In response to the sovereign requirement for national standards the National Measurement Institute, Australia (NMIA) has developed a measuring system using isotope dilution mass spectrometry (IDMS) to certify forensic aqueous ethanol solutions. NMIA participated in an international study, CCQM-K27, organized under the auspices of the International Committee for Weights and Measures to compare our measuring system with the techniques being used for certifying aqueous ethanol solutions in other metrology institutes. This comparison provided objective evidence that the measuring system developed was fit for the purpose of certifying aqueous ethanol solutions that ranged in concentration from 0.8 mg/g to 120 mg/g. A complete measurement uncertainty budget is presented and shows that the largest contribution to measurement uncertainty was from method precision followed by the contribution from the calibration solution. The fundamental technology of the measuring system was gas chromatography of the aqueous ethanol solutions using porous layer open tubular columns, and this effectively produced peak area measurements with both GC/MS and GC-FID. It was found that deactivation of the chromatographic system was critical for obtaining reproducible peak shapes and peak area measurements. A range of measuring systems, all using this gas chromatographic technology, was investigated. When conditions were carefully controlled there was no difference in measurement results from GC-IDMS, GC/MS or GC-FID. There was also no difference in results from on-column or split injection systems. A significant issue with the IDMS system was the fragmentation of 13C2-ethanol to produce an ion with the same mass as the molecular ion of ethanol which lead to isobaric interference; careful measurement of this fragmentation ratio was necessary to calculate accurate mass fraction values. NMIA has adopted the GC-IDMS split measuring system to certify aqueous ethanol solutions for Australian legal requirements since this measuring system provided higher analytical specificity than GC-FID, accuracy that was fit for purpose and was operationally less stringent than on-column techniques.

An international comparability study on quantification of total methyl cytosine content.

Various methods have been developed for quantitative analysis of DNA methylation. However, there is currently no reference analysis system regarding DNA methylation with which other analytical approaches can be compared and evaluated. A standard measurement system that includes reference methods and reference materials may improve comparability and credibility of data obtained from different analytical environments. In an effort to establish a standard system for measurement of DNA methylation, the Korea Research Institute of Standards and Science (KRISS) coordinated an international comparison study among different national metrology institutes. An initial stage of the study involved an intercomparison regarding quantitative measurement of total methyl cytosine contents in artificially constructed DNA samples. The measurement principle involved measurement of dNMP contents following enzymatic hydrolysis of DNA samples. Results of the study showed good comparability among four of five participants and close agreement with reference values assigned by the coordinating laboratory. Conflicting data from one participant may have resulted from incomplete hydrolysis of samples due to use of insufficient amounts of enzymes. These results indicate that comparable and accurate results can be obtained from different measurement environments if digestion conditions are controlled appropriately and valid calibration systems are employed.

Complete equation for the measurement of organic molecules using stable isotope labeled internal standards, exact matching, and mass spectrometry.

Highly accurate measurements of the amount of substance of organic molecules in a test material can be obtained using exactly matched calibration solutions and internal standards that are labeled with stable isotope atoms by measuring the amount ratio of analyte to internal standard using mass spectrometry. Estimating the uncertainty of quantitative measurements of organic molecules is a means of evaluating accuracy and of establishing traceability to the International System of Units (SI) and requires a measurement function that fully describes the measuring system. This paper presents the derivation of the equation (measurement function) that describes this complete measurement after the internal standard has equilibrated with the test material matrix. It is similar to the equation for inorganic measurements using isotope dilution techniques, but potential biases during chemical processing arising from whole organic molecule analysis compared to inorganic atomic analysis required greater investigation of the yield factors that occur during organic molecule measurements. In the new equation, a series of ratios of proportionality factors are used to relate the amount of substance in a test material to chromatographic peak area ratios corresponding to mass spectrometer ion current ratios. All the proportionality factors are grouped together to define a measuring system factor F(X), the value of which is determined by the fundamental chemical processes affecting the yields of analyte, internal standard, and reference standard of the analyte in the measurement process. Any factors in the measurement process that affect the mole ratio of analyte to internal standard in the calibration solution differently from the test solution will result in a nonunity value for F(X) and a proportional bias to the measurement, and in this way F(X) represents the concept of recovery of the amount ratio of analyte to internal standard. Thus highly accurate measurements require F(X) or its constituent factors to be evaluated. In addition, the uncertainty in the evaluation of F(X) or of its constituent factors must be included in a complete uncertainty estimation of the analytical procedure. The many different permutations of proportionality factor ratios that may result in a unity value of F(X) are discussed resulting in a case for evaluating F(X) rather than the more common practice of evaluating individual factors for each major stage of the measurement procedure. Since the new measurement function describes the complete chemical process that constitutes the measurement, traceability to the SI is assured when all factors in the function are measured traceably and have their associated uncertainty estimated correctly. Ignoring F(X) would invalidate traceability to the SI and would prevent a complete estimation of measurement uncertainty.