A set of three plasmid DNA calibration solutions bearing a ruminant-specific DNA fragmentCertified Reference Materials: ERM-AD482a,ERM-AD482b, ERM-AD482c

摘要

This report describes the preparation and characterisation of a set of three plasmid solutions, ERM-AD482a, ERM-AD482b and ERM-AD482c. The material was produced in accordance with ISO Guide 34:2009 [1]. A DNA fragment specific for the identification of ruminant meat was cloned into a pUC18 vector to construct the pIRMM-0103 plasmid. The nucleic acid sequence of the entire pIRMM-0103 plasmid was determined by dye terminator cycle sequencing applying the primer walking method on the entire plasmid. The plasmid was put into a solution and its concentration was measured by ultraviolet (UV) spectrophotometry. Afterwards this solution was gravimetrically diluted to obtain three different plasmid concentration levels. The plasmid copy number concentration of the three concentration levels were certified by digital quantitative polymerase chain reaction methods (dPCR). In addition, between-unit homogeneity, as well as short-term long-term and freeze-thaw stability was assessed in accordance with ISO Guide 35:2006 [2].The materials are intended for the determination of a cut-off value to discriminate positive samples (containing the ruminant target sequence) from negative samples by quantitative PCR as defined in the Standard Operating Procedure of the EU Reference Laboratory for animal proteins (EURL-AP) according to Commission Regulation No 51/2013 [3, 4].As any certified reference material (CRM), the materials can also be used for control charts or validation studies. The CRM is available as a set of three vials each containing at least 1 mL of plasmid solution. The minimum amount of sample to be used is 4 µL.