Optimierung und quantitative Auswertung eines hochsensitiven Reverse Transkriptase-Aktivitäts Assays

Ziel dieser Arbeit war die Vereinfachung und Optimierung der bekannten hochsensitiven RT-Nachweisverfahren. Hochsensitive RT-Nachweisverfahren bestehen aus der Generierung von cDNA aus einem heteropolymeren Template mit anschließender Amplifikation der entstandenen cDNA mittels PCR. Bei den ersten in der Literatur beschriebenen Verfahren zum hochsensitiven RT-Aktivitätsnachweis mit PCR-Amplifikationsschritt (PERT, Pyra 1994; AmpRT, Heneine 1995) handelt es sich um qualitative Verfahren mit Gel-Elektrophorese und Ethidiumbromid Färbung der PCR-Produkte als Read-out. Von diesen beschriebenen Verfahren wurde die Auswahl des heteropolymeren RNA-Templates und der grundsätzliche Ablauf, sowie einige Überlegungen zur Optimierung übernommen. Im Rahmen dieser Arbeit wurden neue, optimierte Primersequenzen für den RT-Schritt sowie für den PCR-Amplifikationsschritt entworfen. Diese Primersequenzen ermöglichen die Untersuchung der RT-Aktivität im Verlauf des RNA-Templates sowie die Untersuchung des Einflusses von RT-Inhibitoren. Es wurden die üblichen Optimierungsschritte wie MgCl2-Titration, Titration der Primerkonzentration und Variation der Annealing-Temperatur durchgeführt. Um eine Kontamination im Sinne eines Carry-over von PCR-Produkten zu verhindern, wurde ein UNG/dUTP System integriert und in seiner Wirksamkeit überprüft. Zwei verschiedene quantitative Read-out Verfahren wurden verglichen: Die Bestimmung der Menge an entstandenem PCR-Produkt am Ende des PCR-Schrittes stellte sich als arbeitsaufwändig heraus und ermöglichte nur einen geringem linearen Messbereich. Die Real-Time-Quantifizierung zeigte dagegen einen weiten linearen Messbereich und benötigt nach dem PCR-Schritt keine zusätzlichen Arbeitsschritte. Es konnte somit die Überlegenheit des Nachweises mittels Real-Time-PCR im Vergleich zur Endpunkt-Bestimmung gezeigt werden. Zur Real-Time Quantifizierung wurde für die Fluoreszenz-Detektion statt einer Exonuklease-Sonde (TM-PERT, TaqMan-Verfahren, z.B. von Maudru 1998 beschrieben) ein Molecular Beacon verwendet. Auch hier wurden die üblichen Optimierungsverfahren (Sonden-Titration, Einsaatmenge cDNA) durchgeführt. Mit dem entwickelten Verfahren können so geringste Mengen von RT-Aktivität, entsprechend ca. 40 Viruspartikeln mit hoher Kontaminationssicherheit und hoher Spezifität nachgewiesen werden. Es konnte das Ansprechen auf nukleosidische RT-Inhibitoren gesteigert werden, mit der Möglichkeit phänotypische Resistenztests auch gegen AZT durchzuführen. Zahlreiche viel versprechende Anwendungsgebiete des RT-Aktivitäts Nachweises mit dem hier etablierten Test sind jedoch derzeit für die Routineanwendung nicht zugänglich, solange die Probleme durch unspezifische Inhibitoren, wie sie beispielsweise im Blutplasma vorkommen, nicht gelöst sind. Um dieses Nachweisverfahren für die virologische Diagnostik einzuführen, wird ein stabiles und einfaches Aufreinigungsverfahren für RT aus Blutplasma von Patienten benötigt

Exploring the Full Potential of Photocatalytic Carbon Dioxide Reduction Using a Dinuclear Re2Cl2 Complex Assisted by Various Photosensitizers

Photosensitizing units have already been applied to enable light-driven catalytic reduction of CO2 with mononuclear rhenium complexes. However, dinuclear catalytic systems that are able to activate CO2 in a cooperative bimetallic fashion have only rarely been combined with photosensitizers. We here present detailed studies on the influence of additional photosensitizers on the catalytic performance of a dirhenium complex (Re2Cl2) and present correlations with spectroscopic measurements, which shed light on the reaction mechanism. The use of [Ir(dFppy)3] (Ir, dFppy=2-(4,6-difluorophenyl)pyridine)) resulted in considerably faster CO2 to CO transformation than [Cu(xant)(bcp)]PF6 (Cu, xant=xantphos, bcp=bathocuproine). Emission quenching studies, transient absorption as well as IR spectroscopy provide information about the electron transfer paths of the intermolecular systems. It turned out that formation of double reduced species [Re2Cl2]2− along with an intermediate with a Re−Re bond ([ReRe]) can be taken as an indication of multi-electron storage capacity. Furthermore, under catalytic conditions a CO2-bridged intermediate was identified.German Research FoundationDFG http://dx.doi.org/10.13039/501100001659Peer Reviewe

RNA reference materials with defined viral RNA loads of SARS-CoV-2—A useful tool towards a better PCR assay harmonization

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.Peer Reviewe

Dynamical mean-field study of ferromagnetism in the periodic Anderson model

The ferromagnetic phase diagram of the periodic Anderson model is calculated using dynamical mean-field theory in combination with the modified perturbation theory. Concentrating on the intermediate valence regime, the phase boundaries are established as function of the total electron density, the position of the atomic level and the hybridization strength. The main contribution to the magnetic moment stems from the f-electrons. The conduction band polarization is, depending on the system parameters either parallel or antiparallel to the f-magnetization. By investigating the densities of states, one observes that the change of sign of the conduction band polarization is closely connected to the hybridization gap, which is only apparent in the case of almost complete polarization of the f-electrons. Finite-temperature calculations are also performed, the Curie temperature as function of electron density and f-level position are determined. In the intermediate-valence regime, the phase transitions are found to be of second order.Comment: 12 pages, 11 figures, accepted by Phys. Rev.

Simultaneous identification of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis ‒ multicenter evaluation of the Alinity m STI assay

Abstract Objectives Accurate and rapid diagnosis of sexually transmitted infections (STIs) is essential for timely administration of appropriate treatment and reducing the spread of the disease. We examined the performance of the new Alinity m STI assay, a qualitative real-time multiplex PCR test for simultaneous identification of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), and Trichomonas vaginalis (TV) run on the fully automated Alinity m platform. Methods This international, multicenter study evaluated the accuracy, reproducibility, and clinical performance of the Alinity m STI assay compared to commonly used STI assays in a large series of patient samples encountered in clinical practice. Results The Alinity m STI assay identified accurately and precisely single and mixed pathogens from an analytical panel of specimens. The Alinity m STI assay demonstrated high overall agreement rates with comparator STI assays (99.6% for CT [n=2,127], 99.2% for NG [n=2,160], 97.1% for MG [n=491], and 99.4% for TV [n=313]). Conclusions The newly developed Alinity m STI assay accurately detects the 4 sexually transmitted target pathogens in various collection devices across clinically relevant specimen types, regardless of single or mixed infection status

Compositional diversity of rehabilitated tropical lands supports multiple ecosystem services and buffers uncertainties

High landscape diversity is assumed to increase the number and level of ecosystem services. However, the interactions between ecosystem service provision, disturbance and landscape composition are poorly understood. Here we present a novel approach to include uncertainty in the optimization of land allocation for improving the provision of multiple ecosystem services. We refer to the rehabilitation of abandoned agricultural lands in Ecuador including two types of both afforestation and pasture rehabilitation, together with a succession option. Our results show that high compositional landscape diversity supports multiple ecosystem services (multifunction effect). This implicitly provides a buffer against uncertainty. Our work shows that active integration of uncertainty is only important when optimizing single or highly correlated ecosystem services and that the multifunction effect on landscape diversity is stronger than the uncertainty effect. This is an important insight to support a land-use planning based on ecosystem services

Compositional diversity of rehabilitated tropical lands supports multiple ecosystem services and buffers uncertainties

High landscape diversity is assumed to increase the number and level of ecosystem services. However, the interactions between ecosystem service provision, disturbance and landscape composition are poorly understood. Here we present a novel approach to include uncertainty in the optimization of land allocation for improving the provision of multiple ecosystem services. We refer to the rehabilitation of abandoned agricultural lands in Ecuador including two types of both afforestation and pasture rehabilitation, together with a succession option. Our results show that high compositional landscape diversity supports multiple ecosystem services (multifunction effect). This implicitly provides a buffer against uncertainty. Our work shows that active integration of uncertainty is only important when optimizing single or highly correlated ecosystem services and that the multifunction effect on landscape diversity is stronger than the uncertainty effect. This is an important insight to support a land-use planning based on ecosystem services

Improved molecular laboratory productivity by consolidation of testing on the new random-access analyzer Alinity m

Abstract Objectives Automated molecular analyzers have accelerated diagnosis, allowing earlier intervention and better patient follow-up. A recently developed completely automated molecular analyzer, Alinity™ m (Abbott), offers consolidated, continuous, and random-access testing that may improve molecular laboratory workflow. Methods An international, multicenter study compared laboratory workflow metrics across various routine analyzers and Alinity m utilizing assays for human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), high-risk human papillomavirus (HR HPV), and sexually transmitted infection (STI) (Chlamydia trachomatis [CT]/Neisseria gonorrhoeae [NG]/Trichomonas vaginalis [TV]/Mycoplasma genitalium [MG]). Three turnaround times (TATs) were assessed: total TAT (sample arrival to result), sample onboard TAT (sample loading and test starting to result), and processing TAT (sample aspiration to result). Results Total TAT was reduced from days with routine analyzers to hours with Alinity m, independent of requested assays. Sample onboard TATs for standard workflow using routine analyzers ranged from 7 to 32.5 h compared to 2.75–6 h for Alinity m. The mean sample onboard TAT for STAT samples on Alinity m was 2.36 h (±0.19 h). Processing TATs for Alinity m were independent of the combination of assays, with 100% of results reported within 117 min. Conclusions The consolidated, continuous, random-access workflow of Alinity m reduces TATs across various assays and is expected to improve both laboratory operational efficiency and patient care