CERTIFICATION REPORT: The certification of the mass of lambda DNA in a solution Certified Reference Material: ERM®-AD442k

This report describes the processing and certification of genomic lambda deoxyribonucleic acid (DNA) in a solution. ERM®-AD442k is certified for its lambda DNA mass, expressed in ng/µL. The DNA copy number concentration in cp/µL is provided as an indicative value. The material was produced according to ISO Guide 34:2009. A volume of approximately 400 mL of lambda DNA at an approximate concentration of 450 ng/µL was purchased from Promega Corporation and Benelux BV (Madison, USA and Leiden, NL). After homogenising and diluting this solution with TE buffer, 3100 vials of ERM-AD442k were produced. Each ERM-AD442k vial contains a certified DNA mass concentration of 57.53 ng/µL with an expanded combined uncertainty of 1.07 ng/µL. Using two different next generation sequencing (NGS) techniques (i.e. an Illumina platform and the GS Junior platform from Roche), the nucleic acid sequence of the lambda DNA in ERM-AD442k was verified. Non-lambda DNA sequences were identified in this material. The relative proportion of those sequences was estimated by NGS and further quantified by quantitative polymerase chain reaction (qPCR). Traces of the contaminating DNA, mainly coming from the Escherichia coli (E. coli) host used by Promega Corporation and Benelux BV to produce the lambda DNA, were negligible. Between-vial homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Within-vial homogeneity was quantified to determine the minimum sample intake. The certified DNA mass concentration value was obtained by ultraviolet (UV) spectrophotometry and the indicative DNA copy number concentration value by digital PCR (dPCR). The DNA copy number concentration measured by dPCR is consistent with the DNA mass concentration determined by UV spectrophotometry. The measurements were performed according to the scope of accreditation to ISO/IEC 17025:2005. The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence and with adherence to ISO/IEC 17025. Technically invalid results were removed; however no other outliers were eliminated on statistical grounds only. Uncertainties of the certified and indicative values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties relating to possible inhomogeneity and instability, and to characterisation. The material and its certified value are intended to be used for the calibration of DNA quantification methods, quality control and assessment of method performance. As any reference material, it can also be used to establish control charts or in validation studies. The indicative value of the material is, in contrast to the certified value, a value where the uncertainty was deemed too large to allow certification and is therefore less reliable than the certified value. The CRM is available in Axygen maximum recovery polypropylene vial containing a nominal volume of 1.1 mL lambda DNA in solution. The minimum amount of sample to be used is 50 µL for UV spectrophotometry and 68 µL for dPCR. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

CERTIFICATION REPORT A set of three plasmid DNA calibration solutions bearing a porcine-specific DNA fragment Certified Reference Materials: ERM®-AD483a, ERM®-AD483b, ERM®-AD483c

This report describes the preparation and characterisation of a set of plasmid solutions, ERM-AD483a, ERM-AD483b and ERM-AD483c. The materials were produced in accordance with ISO Guide 34:2009 [1]. A DNA fragment specific for the identification of a porcine target was cloned into a pUC18 vector to construct the pIRMM-0104 plasmid. The nucleic acid sequence of the entire pIRMM-0104 plasmid was determined by dye terminator cycle sequencing. The plasmid was eluted in TElow buffer, and its concentration was measured by ultraviolet (UV) spectrophotometry. Afterwards, it was gravimetrically diluted to three different concentration levels. The plasmid copy number concentration of the three concentration levels were certified by digital and droplet digital quantitative polymerase chain reaction methods, dPCR and ddPCR respectively. In addition, between-unit homogeneity, as well as short-term and long-term 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 porcine 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 (EU) No 51/2013 [3, 4]. As any certified reference material (CRM), ERM-AD483 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.JRC.D.2-Standards for Innovation and sustainable Developmen

The preparation and characterization of three solutions of plasmid DNA containing a ruminant-specific fragment with defined copy number concentrations - Reference Materials: IRMM-AD482a,IRMM-AD482b, IRMM-AD482c

This report describes the processing of a set of plasmid solutions, IRMM-AD482a, b and c. The material was produced following ISO Guide 34:2009. A DNA fragment specific for the identification of ruminant meat were cloned into a pUC18 vector to construct the pIRMM-0103 plasmid. The plasmid was diluted to three different concentration levels. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The materials are intended for the determination of a cut-off value to discriminate positive samples from negative samples by quantitative PCR. As any reference material, the materials can also be used for control charts or validation studies. The RM is available as a set of three plastic tubes containing 1 mL of plasmid solution. The minimum amount of sample to be used is 4 μL.JRC.D.2-Standards for Innovation and sustainable Developmen

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

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.JRC.D.2-Standards for Innovation and sustainable Developmen

DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials

The value assignment for properties of six certified reference materials (ERM-AD623a-f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per µL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (BioRad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as defined parameter in the QuantaSoft software version 1.3.2.0 (BioRad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with BioRad’s new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration.JRC.D.2-Standards for Innovation and sustainable Developmen