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.

Storage Stability of Solutions of DNA Standards.

High accuracy, reliability, and reproducibility of genetic analyses in various applications require optimized and validated protocols and standards. Optimal procedures for storing the genetic material extracted from biological samples are equally important. In this study, we investigated the stability of dilute (4000 cp/μL, nominal concentration, equivalent to 0.

Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy.

There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes.

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.

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994.

DNA methylation ratio variability may impede clinical application of cancer diagnostic markers.

Hypermethylation at promoter regions of tumour suppressor genes is diagnostic for many cancers. Many genomic regions that may be the targets for clinical diagnostic assays have been identified through use of measuring systems reliant on bisulphite conversion, but few of these promising markers are in clinical use. The comparability of a widely used DNA methylation measuring system involving bisulphite conversion was evaluated by supplying three experienced centres with methylated DNA reference material mixtures that were independently prepared and characterised by mass spectrometry and high-pressure liquid chromatography.

Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction.

Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs).

A study of comparability in amplified fragment length polymorphism profiling using a simple model system.

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated.

Evaluation of a DNA fingerprinting method for determining the species origin of meats.

This study describes an investigation into the use of a PCR-RFLP technique as a routine analytical tool for species testing. The technique was used to generate DNA fingerprints for 22 animal species by amplifying a 359 bp region within the cytochrome b gene and digesting the amplified product using Hae III and Hinf I. All species could be discriminated using the two restriction enzymes with the exception of kangaroo and buffalo.

Identification of fish species using random amplified polymorphic DNA (RAPD).

The random amplified polymorphic DNA (RAPD) method was investigated as a potential fish species identification method. One hundred and sixteen specimens from eight species of fish were analysed. The eight species tested were barramundi, Nile perch, john dory, mirror dory, silver dory, spikey oreo, warty oreo and smooth oreo.

Inhibitory effects of enrichment media on the Accuprobe test for Listeria monocytogenes.

During an evaluation of the Accuprobe kit for the detection of Listeria monocytogenes, some of the enrichment media used were found to interfere with the test. Microscopic examination during the lysis step of the test revealed that media containing high salt greatly reduced or prevented cell lysis. This prevented the probe from binding to the cellular RNA, resulting in false-negative results.