Helen C. Parkes

Identification of the species of origin of raw and cooked meat products using oligonucleotide probes

A simple assay suitable for the routine determination of species composition in admixtures of meat is described. A nonradioactive slot blot hybridisation assay using species-specific oligonucleotide probes has been developed and applied to the species identification of rabbit, sheep, pork, beef and goat meats. Clear species discrimination was demonstrated even between the closely related ruminants goat and sheep. The probes were shown to identify species present in both raw and commercially cooked and canned products (e.g. petfood). The potential for semi-quantitation of species in admixture was demonstrated to a detection limit of less than 2.5% adulteration. This DNA assay targets intracellular DNA and can therefore overcome the potential problem of blood and plasma drip contamination which has led to uncertainty when using soluble immunoassays directed towards soluble plasma protein.

Evaluation of LabChipTM technology for GMO analysis in food

Abstract Ongoing technological advances permit the development of new scientific strategies for bio-analysis. The work described here sought to compare a traditional agarose slab gel electrophoresis method combined with digital image analysis against a new microfluidic capillary electrophoresis system (LabChipTM, Agilent Technologies) to assess their relative performance in the quantitative analysis of polymerase chain reaction (PCR) products. The comparative strategy exploited methodology for providing quantitative genetically modified organism (GMO) determinations in food samples and demonstrated that the LabChipTM system offered improvements in quantitation accuracy, objectivity and ease of use.

Comparison of small-scale methods for the rapid extraction of plant DNA suitable for PCR analysis

We present results from a comparison of six methods for rapid DNA extraction from leaf and other plant tissues. We have used samples from six plant species in our study, including both crop species and their wild relatives. The success of the methods is assessed by PCR of the DNA using conserved primers, and the applicability of the different methods to particular species and tissues is assessed. The speed, reliability, convenience, and potential for further improvement of the methods are also discussed.

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

ABSTRACT Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

Personal monitoring of exposure to genetically modified microorganisms in bioaerosols: Rapid and sensitive detection using PCR

Abstract E. coli XL1-B cells, genetically modified to contain the gene for the commerically important food-processing enzyme, bovine chymosin, were aerosolised in growth media to simulate a breach of containment. Aerosols were generated in a well-characterised bioaerosol test chamber, and sampled using two commonly employed workplace aerosol samplers—the Cyclone static “area” sampler, and the IOM Personal Inspirable Aerosol Sampler. PCR-based detection procedures were developed for the specific, sensitive, and rapid, detection and discrimination of both captured aerosolised genetically modified and unmodified E. coli cells. The IOM personal sampler proved to be more useful than the cyclone sampler for aerosol capture and subsequent analysis using the PCR procedure. it allowed an apparent lower limit of detection of an aerosol containing 1.7 × 10 4 cells m −3 , with results being obtained within 4–5 h after sample collection.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals (‘measurement uncertainties’) were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials. The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comit? Consultatif pour la Quantit? de Mati?re (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium). The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), T?BITAK UME (Turkey) and VNIIM (Russian Federation). The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand). Good agreement was observed between the reported results of eleven participants. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine

BACKGROUND Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

BETTER MEASUREMENT FOR IMPROVED DIAGNOSIS AND MANAGEMENT OF ALZHEIMER'S DISEASE: UPDATE ON THE EMPIR NEUROMET PROJECT

The development of novel therapies for Alzheimer’s Disease (AD) is constrained by the lack of available methods for preclinical diagnosis, despite extensive research on biomarker identification. He ...