Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-017-0206-0) contains supplementary material, which is available to authorized users

Badenian discoasters from the section in Lenart (Northeast Slovenia, Central Paratethys)

In Slovenske gorice, south of Lenart, a 20 m profile of Middle Miocene strata has been exposed. During previous research numerous discoasters have been found among other coccoliths. In Slovenia Miocene discoasters have only been found in Badenian sedimentsin Slovenske gorice and they are particularly useful for paleoecological reconstructions.Additional samples were taken from three selected sections in the middle part of the profile, targeting strata with the greatest abundance of discoasters. In two of the three examined sections 9 species of discoasters were identified, the most abundant being D.exilis and D. variabilis. Even though warm water species were found in samples from all three sections, discoasters only occured in two short intervals. This pattern is not a result of temperature changes and is in our opinion connected with the changes in nutrient levelsof seawater

Comparison of Biocoenosis with Thanatocoenosis from Adriatic, between the peninsula Istria and the river Po

The comparison of biocoenosis and thanatocoenosis at three observational stations, west of the peninsula Istria, in littoral of the Adriatic sea, has shown a suprisingly low similarity between them. The difference seems to be due to ecological changes during the deposition of thanatocoenosis (approximately in thelast hundert years). These ecological changes can not be studied in thanatocoenosis because of a very high degree of bioturbation that destroyed stratification and mixed all the skeletal particles. Lateral transport of skeletal particles seems to be of minor importance

Oligocene sawfish from Breg near Čeplje, Slovenia

In paper a 168 mm long rostral tooth of a cartilaginous fish – sawfish from Breg near Čeplje is decsribed. In its proximal part the tooth is thick and broad, and in the distal part narrow, very flattened and thinned. It was found in greenish-grey Oligocene calcareous sandstone containing some mica. This is the first recorded find of a cartilaginous fish of genus Pristis in Slovenia. The calcitic nannoplankton in the beds at Breg permits their attribution to Middle Kiscellian of the Lower Oligocene

The »Carniolan crab« from Middle Miocene – Badenian beds in the Lipovica quarry above Bri{e, Slovenia

Examined were remains of »Carniolan crab« of the decapod species Tasadia carniolica (Bittner, 1884) from Middle Miocene – Badenian beds in the Lipovica quarry above Briše.The species was first established in Slovenia in a locality between Tunjice and Kamnik.Individual modest remains were registered also in other Slovenian localities. Later the species was found also elsewhere in the Central Paratethys and in the region of the Borealbioprovince

Triassic and Jurassic beds in Krim Mountain area (Slovenia)

The Krim Mountain and its surroundings are characterized by Upper Triassic to Middle Jurassic rocks, which were deposited on the northern margin of the Dinaric Carbonate Platform. Upper Triassic beds are represented by Main dolomite that exhibits supra- to subtidal Lofer facies. The uppermost Triassic is characterized by approximately 40 m thick horizon of dolomitic breccia. Upper Triassic beds pass gradually into Lower Liassic dolomitic breccia, coarse-grained dolomite and micritic limestone. Presence of dolomitic breccias and absence of supra-intertidal sedimentary structures indicate sea-level rise. Middle Liassic beds consist of oolitic-oncolitic and lithiotid limestones deposited in alternating restricted lagoonal and open shallow-water environment. Upper Liassic beds are characterized by oolitic-oncolitic limestones, bituminous dolomitized limestones and dolomitic breccia deposited in high-energy shallow-water environment. Middle Jurassic beds consist of oolitic, oolitic-oncolitic and micritic limestones, formed predominantly in high-energy subtidal environment

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

Background Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. Methods To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. Results dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. Conclusions TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB