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.

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.

Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction.

Digital polymerase chain reaction (dPCR) has the potential to enable accurate quantification of target DNA copy number provided that all target DNA molecules are successfully amplified. Following duplex dPCR analysis from a linear DNA target sequence that contains single copies of two independent template sequences, we have observed that amplification of both templates in a single partition does not always occur. To investigate this finding, we heated the target DNA solution to 95 °C for increasing time intervals and then immediately chilled on ice prior to preparing the dPCR mix.