Development of new RT-PCR assays for the specific detection of BA.2.86 SARS-CoV-2 and its descendent sublineages.

The SARS-CoV-2 BA.2.86 variant, also known as Pirola, has acquired over 30 amino acid changes in the Spike protein, evolving into >150 sublineages within ten months of its emergence.

Assessment of the clinical and analytical performance of three Seegene Allplex SARS-CoV-2 assays within the VALCOR framework.

The coronavirus disease 2019 (COVID-19) pandemic demonstrated the need for accurate diagnostic testing for the early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pandemic has ended, accurate assays are still needed to monitor viral spread at national levels and beyond through population and wastewater surveillance. To enhance early detection, SARS-CoV-2 assays should have high diagnostic accuracy and should be validated to assure accurate results.

Proficiency of European GMO control laboratories to quantify MON89788 soybean in a meat pâté matrix.

GMO control laboratories in the EU routinely monitor the presence and content of genetically modified organisms (GMOs) in food and feed products collected from the EU market. As the vast majority of GMOs comprize genetically modified plants, most control samples have a plant-based origin. For the first time, a pilot proficiency test was organised requiring the analysis of GMOs in a meat matrix.

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron.

Single and multi-laboratory validation of a droplet digital PCR method.

The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control. Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods.

A qualitative RT-PCR assay for the specific identification of the SARS-CoV-2 B.1.1.529 (Omicron) Variant of Concern.

Objectives: The aim of this study was to develop a RT-PCR assay for the specific detection of the SARS-CoV-2 Omicron Variant of Concern (VOC) as a rapid alternative to sequencing.

Methods: A RT-PCR was designed in silico and then validated using characterised clinical samples containing Omicron (both BA.1 and BA.

Expression of GM content in mass fraction from digital PCR data.

Nowadays the quantification of the content of genetically modified (GM) constituents in food or feed products is performed by using either quantitative real-time PCR (qPCR) or digital PCR (dPCR). The latter is increasingly used. Therefore, experimental protocols for the quantification of 52 GM events authorised in the EU have been converted into a digital format and minimum performance characteristics for dPCR methods are detailed.

Validation by collaborative trial of a method for the determination by GC-MS and LC-MS/MS of boar taint marker compounds in pork tissue.

Meat from male pigs may develop an off-flavour, commonly known as boar taint. Castration of male piglets prevents the potential formation of off-flavour. In the suggested method, three marker compounds for boar taint (skatole, androstenone and indole) are quantified in pork fat by isotope dilution gas chromatography mass spectrometry (GC-MS) or by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS).

Can official control laboratories quantify reliably fipronil in eggs? Evidence from a proficiency testing round.

A proficiency testing (PT) round dedicated to assess the competence of European Union (EU) National Reference Laboratories (NRLs), EU Official Control Laboratories (OCLs) and OCLs from associated countries for the determination of the insecticide fipronil in eggs is described. The content of the target analytes (sum of fipronil plus its metabolite fipronil sulfone, expressed as fipronil in the PT test material) was set around the regulated Maximum Residue Level (MRL) set by EU legislation. The PT was organised by the European Commission's (EC) Directorate General (DG) Joint Research Centre (JRC) - in agreement with the EC DG for Health and Food Safety (SANTE) - following a request by the Belgian Authorities.

Analytical method for the trace determination of esterified 3- and 2-monochloropropanediol and glycidyl fatty acid esters in various food matrices.

Fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPDEs), of 2-monochloro-1,3-propanediol (2-MCPDEs) and of 2,3-epoxy-1-propanol or glycidol (GEs), which are considered to be deleterious to human health, may occur in a broad variety of food samples. A proper risk assessment of those substances requires the availability of robust occurrence data; in this respect concerns have been raised regarding the reliability of results obtained with the currently available methods to determine those substances in processed food. This article presents an indirect analytical procedure for the simultaneous determination of 3-MCPDEs, 2-MCPDEs and GEs in a wide variety of food products after extraction by pressurised liquid extraction (PLE) and determination by gas chromatography mass-spectrometry (GC-MS).

Single-laboratory validation of a saponification method for the determination of four polycyclic aromatic hydrocarbons in edible oils by HPLC-fluorescence detection.

An analytical method is reported for the determination of four polycyclic aromatic hydrocarbons (benzo[a]pyrene (BaP), benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and chrysene (CHR)) in edible oils (sesame, maize, sunflower and olive oil) by high-performance liquid chromatography. Sample preparation is based on three steps including saponification, liquid-liquid partitioning and, finally, clean-up by solid phase extraction on 2 g of silica. Guidance on single-laboratory validation of the proposed analysis method was taken from the second edition of the Eurachem guide on method validation.

Aflatoxin measurements: how HPLC methods have evolved over the last 20 years?

The Institute for Reference Materials and Measurements (IRMM) has provided high quality certified reference materials for decades. Certified reference materials for mycotoxin analysis have been available from the IRMM since 1985. Since then, IRMM has generally characterised reference materials by interlaboratory studies with expert laboratories.

Comprehensive molecular system to study the presence, growth and ochratoxin A biosynthesis of Penicillium verrucosum in wheat.

Based on the sequence of the ochratoxin A polyketide synthase gene (otapksPV), a polymerase chain reaction (PCR) system for the specific detection of Penicillium verrucosum in wheat has been developed. In a further approach, a real-time PCR system has been applied to determine the growth kinetics of P. verrucosum in wheat at cell numbers above 10(3) colony-forming units (cfu) ml(-1).

Molecular and chemical monitoring of growth and ochratoxin a biosynthesis ofP. verrucosum in wheat stored at different moisture conditions.

The growth and ochratoxin A producing activity at the phenotypical and molecular level ofP. verrucosum in wheat stored at different relative moisture conditions have been followed. Growth has been measured by determination of colony forming units (cfu).

Determination of molar absorptivity coefficients for major type-B trichothecenes and certification of calibrators for deoxynivalenol and nivalenol.

This paper presents results from the European Commission-funded project Doncalibrant, the objective of which was to produce calibrators with certified mass fractions of the Fusarium toxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-Ac-DON), 15-acetyldeoxynivalenol (15-Ac-DON), and nivalenol (NIV), in acetonitrile. The calibrators, available in ampoules, were sufficiently homogeneous, with between-bottle variations (s (bb)) of less than 2%. Long-term stability studies performed at four different temperatures between -18 and 40 degrees C revealed no significant negative trends (at a confidence level of 95%).

Simultaneous determination of type A-& B-trichothecenes and zearalenone in cereals by High Performance Liquid Chromatography - Tandem Mass Spectrometry.

A rapid quantitative method for the simultaneous determination of the majorFusarium mycotoxins nivalenol, deoxynivalenol, fusarenon-X, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, T-2 toxin and zearalenone in maize and wheat was developed. Raw extracts (acetonitrile/water 84/16) are cleaned-up with MycoSep(®) columns., Chromatographic separation and end determination is carried out by HPLC-APCI-MS/MS.

Performance of new clean-up column for the determination of ochratoxin A in cereals and foodstuffs by HPLC-FLD.

The performance of the newly developed Mycosep 229 Ochra and Multisep 229 Ochra clean-up columns for ochratoxin A (OTA) determination was evaluated. OTA was subsequently analysed using RP-HPLC with fluorescence detection. Recoveries for frequently contaminated commodities, like cereals, red wine, raisins and green coffee, were estimated.

Rapid simultaneous determination of major type A- and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry.

A novel method for the simultaneous determination of the Fusarium mycotoxins nivalenol, deoxynivalenol, fusarenon-X, 3-acetyl-deoxynivalenol, the sum of 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, diacetoxy-scirpenol, HT-2 toxin, T-2 toxin and zearalenone in maize has been developed using gradient RP-LC with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS). Swift clean-up of maize samples was performed with MycoSep #226 columns. Quantification of zearalenone was performed with zearalanone as internal standard (IS), while no IS was used for the trichothecenes.

Molecularly imprinted polymer-assisted sample clean-up of ochratoxin A from red wine: merits and limitations.

A new analytical method for the determination of the carcinogenic mycotoxin ochratoxin A (OTA) in red wines has been developed involving a two-dimensional solid-phase extraction (SPE) clean-up protocol on C18-silica and a target-selective molecularly imprinted polymer (MIP). Prior removal of the interfering acidic matrix compounds by C18 solid-phase extraction was crucial for a successful clean-up as direct sample loading onto the MIP led to poor recoveries. The combined solid-phase extraction protocol afforded extracts suitable for sensitive ochratoxin A quantification by HPLC-fluorescence detection.

Determination of B-trichothecenes in wheat by post column derivatisation liquid chromatography with fluorescence detection (PCD-HPLC-FLD).

B-trichothecenes are one of the most common contaminants of cereals in Europe. Therefore, the use of fast and accurate methods is necessary to measure contamination levels and observe regulatory limits.At the moment, mostly gas chromatographic (GC) methods are used but HPLC-UV methods are also employed.

Synthesis of deoxynivalenol-glucosides and their characterization using a QTrap LC-MS/MS.

Deoxynivalenol (DON)-glucosides were successfully synthesized in a two-step reaction from 1-β-Bromo-1-deoxy-2,3,4,6-tetra-O-acetyl-α-D-gluco-pyranose and 3-Acetyl-DON or 15-Acetyl-DON. After purification of the reaction products, the mycotoxin conjugates were for the first time characterized by means of a triple quadrupole mass spectrometer in combination with a linear ion trap. Due to different fragmentation behaviour it was also possible to distinguish between the two glucosides.