Suchergebnisse

On 7 October 2015 the High Court of Australia unanimously allowed the appeal on D'Arcy v. Myriad Genetics Inc and ordered that claims 1, 2 and 3 of Australian Patent No 686004, entitled "In vivo mutations and polymorphisms in the 17q-linked breast and ovarian cancer susceptibility gene", be revoked.The High Court's judgment overturned the decisions of Justice Nicholas of the Federal Court, at first instance, and the Full Federal Court. This case note provides an overview of the High Court's decision and discusses its meaning and implications for patenting isolated nucleic acids in Australia.

Over the past two decades, several computational methods have been proposed to predict how missense mutations can affect protein structure and function, either by altering protein stability or interactions with its partners, shedding light into potential molecular mechanisms giving rise to different phenotypes. Effectively and efficiently predicting consequences of mutations on protein–nucleic acid interactions, however, remained until recently a great and unmet challenge. Here we report an updated webserver for mCSM–NA, the only scalable method we are aware of capable of quantitatively predicting the effects of mutations in protein coding regions on nucleic acid binding affinities. We have significantly enhanced the original method by including a pharmacophore modelling and information of nucleic acid properties into our graph-based signatures, considering the reverse mutation and by using a refined, more reliable data set, based on a new release of the ProNIT database, which has significantly improved the reliability and applicability of the methodology. Our new predictive model was capable of achieving a correlation coefficient of up to 0.70 on cross-validation and 0.68 on blind-tests, outperforming its previous version. The server is freely available via a user-friendly web interface at: http://structure.bioc.cam.ac.uk/mcsm_na. ; Jack Brockhoff Foundation [JBF 4186, 2016 to D.B.A.]; Newton Fund RCUK-CONFAP Grant awarded by The Medical Research Council (MRC) and Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) [MR/M026302/1 to D.B.A. and D.E.V.P.]; National Health and Medical Research Council of Australia [APP1072476 to D.B.A.]; Victorian Life Sciences Computation Initiative (VLSCI), an initiative of the Victorian Government, Australia, on its Facility hosted at the University of Melbourne [UOM0017]; Centro de Pesquisas Rene Rachou (CPqRR/FIOCRUZ Minas), Brazil [to D.E.V.P.]; Department of Biochemistry and Molecular Biology, University of Melbourne [to D.B.A.]. Funding for open access charge: MRC.

In this review, we summarize the current status of nucleic acid and antigen testing required for diagnosing SARS-CoV-2 infection and COVID-19 disease. Nucleic acid amplification (NAAT) and antigen-detection (Ag) tests occupy a critically important frontline of defense against SARS-CoV-2 in clinical and public health settings. In early stages of this outbreak, we observed that identifying the causative agent of a new illness of unknown origin was greatly accelerated by characterizing the nucleic acid signature of the novel coronavirus. Results from nucleic acid sequencing led to the development of highly sensitive RT-PCR testing for use in clinical settings and to informing best practices for patient care, and in public health settings to the development of strategies for protecting populations. As the current COVID-19 pandemic has evolved, we have seen how NAAT performance has been used to guide and optimize specimen collection, inform patient triage decisions, reveal unexpected clinical symptoms, clarify risks of transmission within patient care facilities, and guide appropriate treatment strategies. For public health settings during the earliest stages of the pandemic, NAATs served as the only tool available for studying the epidemiology of this new disease by identifying infected individuals, studying transmission patterns, modeling population impacts, and enabling disease control organizations and governments to make challenging disease mitigation recommendations to protect the expanding breadth of populations at risk. With time, the nucleic acid signature has provided the information necessary to understand SARS-CoV-2 protein expression for further development of antigen-based point-of-care (POC) diagnostic tests. The advent of massive parallel sequencing (ie, next generation sequencing) has afforded the characterization of this novel pathogen, informed the sequences best adapted for RT-PCR assays, guided vaccine production, and is currently used for tracking and monitoring SARS-CoV-2 variants.

In der vorliegenden Arbeit wird die Untersuchung neuer methodischer Ansätze für die Analyse genetisch veränderter Organismen (GVO) in Lebens- und Futtermitteln beschrieben. Der Bedarf für die Entwicklung alternativer GVO-Nachweismethoden ergibt sich aus den Rechtsvorschriften bezüg¬lich der Rückverfolgbarkeit von GVO in der Europäischen Union sowie der fortschreitenden Zu¬nahme von GVO in Europa. Die durchgeführten Arbeiten hatten grundsätzlich zwei Schwerpunkte. Einerseits wurde ein Verfahren der Gesamtgenomamplifikation, die multiple displacement amplification, für die An¬reicherung von Proben-DNA eingesetzt und hinsichtlich ihrer Eignung für die GVO-Analyse unter¬sucht. Genomische DNA verschiedener genetisch veränderter Maislinien wurde mit dieser Methode amplifiziert. Die resultierende DNA wurde hinsichtlich ihrer Ausbeute und Qualität beurteilt. In Real-Time PCR (Polymerasekettenreaktion)-Analysen wurde das Verhalten der ampli¬fizierten DNA mit dem der Ausgangs-DNA verglichen. Zudem wurde die Gleichmäßigkeit der Ver¬viel¬fältigung verschiedener Genom-Abschnitte geprüft. Unabhängig von der Menge eingesetzter genomischer DNA wurde eine konstante Ausbeute hochmolekularer Mais-DNA generiert, die sich in der Real-Time PCR identisch zur Ausgangs-DNA verhielt. Die Amplifikation verschiedener Genom¬abschnitte war weitgehend gleichmäßig. Die mittels multiple displacement amplification vervielfachte DNA ist für die qualitative GVO-Analyse geeignet und kann zur Anreicherung von Proben-DNA oder als Referenzmaterial ver¬wendet werden. Das zweite Teilziel der Arbeit war die Entwicklung eines DNA-Nachweisverfahrens für die parallele GVO-Detektion als Alternative zur PCR. Die Methodik sollte auf den Ergebnissen des ersten Teils aufbauen, indem die Gesamtgenomamplifikation als Grundlage für die Bereitstellung von Proben-DNA genutzt werden sollte. Es wurde eine neue Strategie für die Sequenz-spezifische DNA-Detektion entwickelt, bei der eine Kombination aus direkter Hybridisierung genomischer DNA, immunologischer Detektion und Signal-Amplifikation mittels einer uniformen PCR ein¬gesetzt wird. Die Detektion und Visualisierung basiert auf dem Prinzip der aus dem Bereich der Protein-Analytik stammenden Real-Time Immuno-PCR. Nach dem Aufbau einer geeigneten Methodik und der Optimierung einzelner Arbeitsschritte wurde die Leistungsfähigkeit der Methode für die Detektion ver¬schiedener Zielmoleküle untersucht. Die Ergebnisse belegen erstmals den Sequenz-spezifischen, selektiven Nachweis von DNA mit der Real-Time Immuno-PCR, die bisher ausschließlich für den Nachweis von Antigenen verwendet wurde. Sowohl synthetische Oligo¬nukleotide als auch PCR-ampli¬fizierte Proben-DNA ließen sich mit und ohne Anwesenheit von Hintergrund-DNA mit hoher Messpräzision und Wiederholbarkeit nachweisen. Die Nachweis¬grenze für ein synthetisches DNA-Oligomer unter optimierten Nachweisbedingungen lag bei 6 Attomol, während PCR-ampli¬fizierte DNA in Mischungen mit nicht-komplementären PCR-Amplika bis zu einer Menge von 60 Attomol detektiert wurde. Die hohe Signalstärke der Detektion durch die Signal-Amplifikation mittels PCR war gleichzeitig mit starken Hintergrund-Signalen verbunden, die die Sensitivität der DNA-Detektion in Anwesenheit größerer Mengen unspezifischer DNA verringerten. Versuche mit genomischer Hintergrund-DNA zeigten, dass die Sensitivität der Detektion nicht für den direkten Nach¬weis von Einzelkopie-Sequenzen in komplexer genomischer DNA – wie genetisch veränderter Pflanzen-DNA – ohne eine vorherige Zielsequenz-Amplifikation ausreicht. Ein Einsatz der Methode für andere An¬wendungen ist denkbar, bei denen entweder kleinere Genome (beispielsweise von Bakterien oder Viren) oder mehr¬fach im Genom vorhandene Zielsequenzen untersucht werden. ; The present thesis explores novel approaches for the analysis of genetically modified organisms (GMOs) in foods and feeds. The European legislation related to traceability of GMOs and the constantly increasing number of GMOs in Europe create a demand for the development of alternative GMO detection methods. The focus of this work is on two objectives. On the one hand, multiple displacement amplification, a whole genome amplification technique, was tested for its suitability regarding the enrichment of sample DNA for GMO analysis. Genomic DNA of several genetically modified maize lines was amplified and evaluated in terms of yield and quality. The real-time PCR (polymerase chain reaction) properties of the amplified DNA were assessed in comparison with the original DNA. Additionally, gene representation after multiple displacement amplification was analyzed with respect to a possible amplification bias. Regardless of the amount of input DNA, amplification yielded a constant amount of high molecular weight maize DNA. Real-time PCR performance and gene representation of the amplified DNA were comparable to those of the original DNA. Multiple displacement amplification is a suitable tool for the accumulation of sample DNA and the generation of reference material for qualitative GMO analysis. The second intention of this study was the design and development of a DNA detection method for the parallel detection of GMOs as an alternative to PCR. The approach was to be based on the results of the first part of this work by using the multiple displacement amplification technique for the preparation of sample DNA. A novel strategy for sequence specific DNA detection was developed, applying a combination of direct hybridization of genomic DNA, immunological detection and signal amplification by a uniform PCR. Visualization relies on the principle of real-time immuno-PCR, a technique formerly used for protein detection. A methodological concept was implemented and several experimental steps were optimized. Subsequently, the performance of the method was determined concerning the detection of various target molecules. The obtained results demonstrate for the first time a sequence specific and selective detection of DNA by the real-time immuno-PCR technique. Both synthetic oligonucleotides and PCR amplified sample DNA with or without the presence of background DNA were detected with high precision and repeatability. Under optimized conditions, the limit of detection was 6 attomol for a synthetic DNA oligomer and 60 attomol for a PCR amplified target mixed with non-complementary PCR products. The ampli¬fication power of the PCR resulted in high signal intensities. However, it was coupled with high background signals as well, thereby decreasing the sensitivity of DNA detection in the presence of major amounts of unspecific DNA. When genomic DNA background was included in the reaction, sensitivity proved not to be sufficient for the direct detection of single copy genes in complex genomic DNA – like genetically modified plant DNA – without prior target amplification. Possibly, the method might be employed for other applications than GMO detection, where either less complex genomes or multiple copy genes are analyzed.

Molecular dynamics simulation (MD) is, just behind genomics, the bioinformatics tool that generates the largest amounts of data, and that is using the largest amount of CPU time in supercomputing centres. MD trajectories are obtained after months of calculations, analysed in situ, and in practice forgotten. Several projects to generate stable trajectory databases have been developed for proteins, but no equivalence exists in the nucleic acids world. We present here a novel database system to store MD trajectories and analyses of nucleic acids. The initial data set available consists mainly of the benchmark of the new molecular dynamics force-field, parmBSC1. It contains 156 simulations, with over 120 of total simulation time. A deposition protocol is available to accept the submission of new trajectory data. The database is based on the combination of two NoSQL engines, Cassandra for storing trajectories and MongoDB to store analysis results and simulation metadata. The analyses available include backbone geometries, helical analysis, NMR observables and a variety of mechanical analyses. Individual trajectories and combined meta-trajectories can be downloaded from the portal. The system is accessible through http: //mmb.irbbarcelona.org/BIGNASim/. Supplementary Material is also available on-line at http://mmb. irbbarcelona.org/BIGNASim/SuppMaterial/. ; Spanish Ministry of Science [BIO2012-32868, SEV-2011-00067, TIN2012-34557]; Catalan Government [2014-SGR-134, 2014-SGR-1051]; Institut Català de Recerca I Estudis Avanc¸ats, ICREA Academia [to M.O.], Instituto de Salud Carlos III-Instituto Nacional de Bioinformática [PT13/0001/0019, PT13/0001/0028]; European Research Council [ERC SimDNA]; European Union, H2020 programme [Elixir-Excellerate: 676559; BioExcel: 674728, MuG: 676566]; PEDECIBA and SNI (ANII, Uruguay) [to P.D.D.]. Funding for open access charge: European Union [MuG: 676566]. ; Peer Reviewed ; Postprint (published version)

Molecular dynamics simulation (MD) is, just behind genomics, the bioinformatics tool that generates the largest amounts of data, and that is using the largest amount of CPU time in supercomputing centres. MD trajectories are obtained after months of calculations, analysed in situ, and in practice forgotten. Several projects to generate stable trajectory databases have been developed for proteins, but no equivalence exists in the nucleic acids world. We present here a novel database system to store MD trajectories and analyses of nucleic acids. The initial data set available consists mainly of the benchmark of the new molecular dynamics force-field, parmBSC1. It contains 156 simulations, with over 120 of total simulation time. A deposition protocol is available to accept the submission of new trajectory data. The database is based on the combination of two NoSQL engines, Cassandra for storing trajectories and MongoDB to store analysis results and simulation metadata. The analyses available include backbone geometries, helical analysis, NMR observables and a variety of mechanical analyses. Individual trajectories and combined meta-trajectories can be downloaded from the portal. The system is accessible through http: //mmb.irbbarcelona.org/BIGNASim/. Supplementary Material is also available on-line at http://mmb. irbbarcelona.org/BIGNASim/SuppMaterial/. ; Spanish Ministry of Science [BIO2012-32868, SEV-2011-00067, TIN2012-34557]; Catalan Government [2014-SGR-134, 2014-SGR-1051]; Institut Català de Recerca I Estudis Avanc¸ats, ICREA Academia [to M.O.], Instituto de Salud Carlos III-Instituto Nacional de Bioinformática [PT13/0001/0019, PT13/0001/0028]; European Research Council [ERC SimDNA]; European Union, H2020 programme [Elixir-Excellerate: 676559; BioExcel: 674728, MuG: 676566]; PEDECIBA and SNI (ANII, Uruguay) [to P.D.D.]. Funding for open access charge: European Union [MuG: 676566]. ; Peer Reviewed ; Postprint (published version)

In this report, the authors summarize for the nonscientific community how current and emerging nucleic acid biosurveillance techniques work, describe their capabilities and limitations, and discuss the promise of emerging technologies.

The first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of C. trachomatis by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the performance of these assays.

Under China's "dynamic zero" COVID-19 policy, Shenzhen required its residents to present a negative nucleic acid testing result within 24 or 48 h to access most public spaces and transit until most recently. The uneven accessibility to testing services could render certain groups vulnerable to mobility disadvantage (e.g., denied access to public transport). Using data of nucleic acid testing services and residents' positioning points, I created a cartogram to capture the spatial distribution of people's activities and that of testing services in Shenzhen. The cartogram indicates that the nucleic acid testing services were spatially concentrated in a way inconsistent with the distribution of people's daily activities. Several girds exhibit high presence of activities but low or no provision of testing services that were necessary for residents to accessing public spaces and transport. The cartogram casts light to potential consequence of regular nucleic acid testing on mobility equality.

The Delta variant (B.1.617.2) has dominated in many countries over the world. Its sudden outbreak in China has led the government to quickly carry out large-scale nucleic acid testing to curb its spread. This qualitative study aims to find the challenges based on empirical evidence from the perspectives of the different groups of people involved in the testing, and further explore possible strategies to improve the efficiency of large-scale nucleic acid testing. Using a phenomenological approach, we selected 35 participants (seven managers, eight health professionals, six community volunteers and 14 residents) by purposive sampling. The interviews were conducted by in-depth semi-structured interviews and the data were analyzed by Colaizzi's seven-step method. Qualitative analysis revealed three main themes: unreasonable and unsafe testing points layout settings, human and medical resources challenges, and potential infection risk. From the different angles, participants all experienced challenges during large-scale nucleic acid testing, making positive planning and adequate preparation important parts of the smooth development of testing. Large-scale nucleic acid testing relies on the cooperation and efforts of all to support containment of the spread of the virus. Local governments should improve their ability to respond to and deal with public health emergencies.

Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situHybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.699.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. ; This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). Antonio Machado was funded by the FCT individual fellowship SFRH/BD/62375/2009. Other fundings came from the FCT Strategic Project PEst-OE/EQB/LA0023/2013, the Project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER, and the project FCOMP-01-0124-FEDER-008991 (RECI/BBB-EBI/0179/2012). Nuno Cerca is an Investigador FCT (Portuguese national funds). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the ...