Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for i

Screening for human immunodeficiency virus type 1 and 2 in a Turkish blood donor population.

In order to calibrate the Super-Kamiokande experiment for solar neutrinomeasurements, a linear accelerator (LINAC) for electrons was installed at thedetector. LINAC data were taken at various positions in the detector volume,tracking the detector response in the variables relevant to solar neutrinoanalysis. In particular, the absolute energy scale is now known with less than1 percent uncertainty

A nucleic acid-based electrochemical biosensor for the detection of influenza B virus from PCR samples using gold nanoparticle-adsorbed disposable graphite electrode and Meldola's blue as an intercalator

WOS: 000292979500025In the presented study, a novel method is introduced that demonstrates the electrochemical detection of influenza B virus based on DNA hybridisation. The detection utilised gold nanoparticles (AuNPs) and Meldola's Blue (MDB), which is utilised as an intercalator label. The developed methodology, combined with a disposable pencil graphite electrode (PGE) and differential pulse voltammetry (DPV), was performed using both synthetic oligonucleotides and polymerase chain reaction (PCR) amplicons. The electrochemical oxidation response of guanine (approximately +0.1 V) and the voltammetric reduction signal of MDB (approximately -0.2 V) were measured before and after hybridisation reactions between a single strand DNA probe and its complementary target strain (synthetic target or denatured PCR samples). Before the immobilisation of the synthetic DNA probe of influenza type B virus, the transducer surface was interacted with AuNPs solution using a simple wet adsorption method. AuNP immobilisation was confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to characterise the recognition surface of the genosensor. After the interaction between the PGE and AuNPs, a thiol-linked DNA probe was immobilised onto the nanoparticle-covered surface. When hybridisation occurred between the probe and its synthetic targets or specific PCR products, the highest MDB signal was observed. The probes were also challenged with equal quantities of non-complementary DNA at the PGE surface for the determination of biosensor selectivity. AuNP-coated electrodes showed high sensitivity and selectivity, specifically in real samples for the detection of the hybridisation reaction. The results obtained in the presented study indicated that the electrode surface area could be enhanced with AuNPs. The detection limit of the genosensor was found to be 54 picomoles for the synthetic target and 3.3 x 10(7) molecules for the real samples (PCR) in 30 mu L of sample volume. Future prospects and analytical performance of the sensor is briefly discussed.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [SBAG-107S163]; Pharmaceutical Sciences Research Centre (FABAL) of Ege University, Faculty of PharmacyEge UniversityThe authors acknowledge financial support from TUBITAK (Projects SBAG-107S163) and the Pharmaceutical Sciences Research Centre (FABAL) of Ege University, Faculty of Pharmacy

Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of lamivudine resistance

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides ("probe") including YMDD (HBV mild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary ("target") sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first time. Features of this protocol are discussed and optimized

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples

Introduction: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Methods: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. Results: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. Conclusion: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories

Evaluation of the results of MOTAKK hepatitis C virus RNA genotyping and hepatitis delta virus external quality assessment programs during 2015-2016

Background/Aims: To evaluate the HCV RNA genotyping and HDV RNA tests that are performed in molecular microbiology laboratories in Turkey as part of a national external quality assessment programme, MOTAKK (Molekuler Tanida Kalite Kontrol) (English translation: Quality control in molecular diagnostics). Materials and Methods: Plasmas having different HCV RNA genotypes were used to prepare HCV genotype control sera. The HDV RNA main stock was prepared from patients with chronic delta hepatitis who had a significant amount of viral load detected, as per the WHO reference materials on viral load studies that were compiled for the purpose of developing HDV RNA control sera. Samples with different viral loads were prepared from this main stock by dilution. The prepared controls were delivered to the registered laboratories. The laboratories carried out the relevant tests and entered their results via the MOTAKK web page. External quality assessment (EQA) reports of the participants were uploaded to the website as well. Results: In total, there were 23 participating laboratories, out of which 20 exclusively performed HCV genotyping, and 15 and 16 only performed HDV RNA in 2015 and 2016, respectively. The success rate of the results of the HCV genotype was 56-96\% in 2015 and 30-95\% in 2016. The tube with a 30\% success rate had a recombinant type of HCV, therefore, it could not be detected in most of the laboratories. The HDV RNA results were evaluated qualitatively. Accordingly, HDV RNA detection rates of participant laboratories were 71-100\% in 2015 and 50-100\% in 2016. Conclusion: This study was the first national external quality control program in Turkey regarding HCV RNA genotyping and HDV RNA in the field of molecular microbiology, and it was implemented successfully