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Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.

• Development and in-house validation of a multiplex RT-qPCR method. • Identification and discrimination of Zika and chikungunya viruses. • Specific and sensitive method applicable to real-life samples. • High coverage of arbovirus strains/isolates. • Testing of the method via a new bioinformatics tool on whole genome sequencing data. The re-emergence and spread of tropical viruses to new areas has raised a wave of concern worldwide. In order to treat patients at an early stage and prevent the diffusion of an outbreak, early diagnosis, and therefore fast and adequate detection, is needed. To this end, a multiplex reverse transcription real-time polymerase chain reaction TaqMan method was designed to detect Zika (ZIKV) and chikungunya (CHIKV) viruses simultaneously. Two methods targeting different genome segments were selected from the literature for each virus. These were adapted for high genome coverage and combined in a four-plex assay that was thoroughly validated in-house. The SCREENED tool was used to evaluate the sequence coverage of the method. The full validation approach showed that the new four-plex method allows the specific and sensitive identification and discrimination of ZIKV and CHIKV in routine samples. The combination of two targets per virus allowing almost 100% coverage of about 500 genomes is shown for the first time. PCR is a reliable user-friendly technique that can be applied in remote areas. Such multiplex methods enable early and efficient diagnosis, leading to rapid treatment and effective confinement in outbreak cases. They may also serve as an aid for surveillance, and the full validation permits easy method-transfer allowing worldwide harmonization. [ABSTRACT FROM AUTHOR]

Background: Viral infection by dengue virus is a major public health problem in tropical countries. Early diagnosis and detection are increasingly based on quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) directed against genomic regions conserved between different isolates. Genetic variation can however result in mismatches of primers and probes with their targeted nucleic acid regions. Whole genome sequencing allows to characterize and track such changes, which in turn enables to evaluate, optimize, and (re-)design novel and existing RT-qPCR methods. The immense amount of available sequence data renders this however a labour-intensive and complex task. Results: We present a bioinformatics approach that enables in silico evaluation of primers and probes intended for routinely employed RT-qPCR methods. This approach is based on analysing large amounts of publically available whole genome data, by first employing BLASTN to mine the genomic regions targeted by the RT-qPCR method(s), and afterwards using BLASTN-SHORT to evaluate whether primers and probes will anneal based on a set of simple in silico criteria. Using dengue virus as a case study, we evaluated 18 published RT-qPCR methods using more than 3000 publically available genomes in the NCBI Virus Variation Resource, and provide a systematic overview of method performance based on in silico sensitivity and specificity. Conclusions: We provide a comprehensive overview of dengue virus RT-qPCR method performance that will aid appropriate method selection allowing to take specific measures that aim to contain and prevent viral spread in afflicted regions. Notably, we find that primer-template mismatches at their 3′ end may represent a general issue for dengue virus RT-qPCR detection methods that merits more attention in their development process. Our approach is also available as a public tool, and demonstrates how utilizing genomic data can provide meaningful insights in an applied public health setting such as the detection of viral species in human diagnostics. [ABSTRACT FROM AUTHOR]

Biotech crops are the fastest adopted crop technology in the history of modern agriculture. The commercialisation of GMO is in many countries strictly regulated laying down the need for traceability and labelling. To comply with these legislations, detection methods are needed. To date, GM events have been developed by the introduction of a transgenic insert (i.e., promoter, coding sequence, terminator) into the plant genome and real-time PCR is the detection method of choice. However, new types of genetic elements will be used to construct new GMO and new crops will be transformed. Additionally, the presence of unauthorised GMO in food and feed samplesmight increase in the near future. To enable enforcement laboratories to continue detecting all GMevents and to obtain an idea of the possible presence of unauthorised GMO in a food and feed sample, an intensive screening will become necessary. A pragmatic, cost-effective, and time-saving approach is presented here together with an overview of the evolution of the GMO and the upcoming needs. [ABSTRACT FROM AUTHOR]