Background: Amplification of viral ribonucleic acid (RNA) by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is the gold standard to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the initial outbreak, strategies to detect and isolate patients have been important to avoid uncontrolled viral spread. Although testing capacities have been upscaled, there is still a need for reliable high throughput test systems, specifically those that require alternative consumables. Therefore, we tested and compared two different methods for the detection of viral PCR products: rRT-PCR and mass spectrometry (MS). Methods: Viral RNA was isolated and amplified from oro- or nasopharyngeal swabs. A total of 22 samples that tested positive and 22 samples that tested negative for SARS-CoV-2 by rRT-PCR were analyzed by MS. Results of the rRT-PCR and the MS protocol were compared. Results: Results of rRT-PCR and the MS test system were in concordance in all samples. Time-to-results was faster for rRT-PCR. Hands-on-time was comparable in both assays. Conclusions: MS is a fast, reliable and cost-effective alternative for the detection of SARS-CoV-2 from oral and nasopharyngeal swabs.
The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its “a” determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the “a” determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of “a” determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.
Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of real-time PCR-based assays with low limits of detection and broad dynamic ranges for quantification. In the present study, the performance of two second-version prototypes of the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with decreased sample input volume and improved genotype inclusivity was investigated. A total of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype 1 [GT1], n ؍ 108; GT2, n ؍ 8; GT3, n ؍ 24; GT4, n ؍ 87; GT5, n ؍ 3; and GT6, n ؍ 2) were processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM), Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4 transcripts containing rare variant sequences were tested. The mean log 10 HCV RNA differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to CAM were ؊0.05, 0.05, ؊0.12, ؊0.10, ؊0.44, and ؊0.29 for patients with GT1, GT2, GT3, GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including isolates with known variants within the 5 untranslated region (G145A, A165T) that were underquantified with CAP/CTM were correctly quantified with the second-version prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA across all HCV genotypes, including specimens with rare polymorphisms previously associated with underquantification.
We compared the analytical and clinical performance of cobas® CT/NG for use on the Cobas® 6800/8800 Systems with the Cobas® 4800 CT/NG Test from urogenital and extragenital specimens in over 12,000 specimens from both male and female subjects in Germany and the United States. The analytical sensitivity was ≤40 EB/ml for Chlamydia trachomatis (CT) and ≤1 CFU/ml for Neisseria gonorrhoeae (NG). Using clinical specimens, the overall percent agreement with the Cobas® 4800 CT/NG Test was >98.5%. Across urogenital specimens, there were 93 discrepant specimens; 76 (93.8%) of 81 CT discrepant specimens were 6800+/4800– and 10 (83.3%) of 12 NG discrepant specimens were 6800+/4800–. Sequencing verified CT results for 45 (61.6%) of 73 samples positive by 6800 and 1 (20%) of 5 positive by 4800. Similarly, 7 (70.0%) of 10 NG samples positive by 6800 and 1 of 2 positive by 4800 were confirmed by sequencing. Among discrepant extragenital specimens (all 6800+/4800–), 7 (50%) of 14 oropharyngeal and 23 (76.7%) of 30 anorectal CT discordant samples were confirmed as CT positive by sequencing; all 8 anorectal and 20 (90.9%) of 22 oropharyngeal NG discordant results were also confirmed as NG positive. In conclusion, Cobas® CT/NG for use on the Cobas® 6800/8800 Systems provides high-throughput automated solutions for sexually transmitted infection (STI) screening programs.
yII-crystallin from calf eye lens consists of two homologous domains, connected by a six-residue linker peptide. In order to study the intrinsic properties of the domains and their mutual stabilization, limited proteolysis was applied. Optimum conditions providing a homogeneous 10-kDa fragment at high yield were pepsin cleavage in 0.1 M NaCl/HCl pH 2.0, in the presence of 3.0 M urea. Determination of the N-terminus and the C-terminal sequence showed that cleavage occurred at the Phe88 -Arg89 peptide bond, giving rise to the complete N-terminal domain including the connecting hexapeptide. The C-terminal part of the polypeptide chain is cleaved to small fragments.Comparing the spectral properties of the isolated N-terminal domain and intact yII-crystallin proved the structure of the fragment to be closely similar to that of the native domain. Small differences in absorbance, fluorescence emission and circular dichroism point to alterations caused by the increase in surface area as a consequence of domain separation. The resistance of the 1 0-kDa fragment toward thermal and alkaline denaturation, as well as unfolding in the presence of urea or guanidine . HCl is decreased, due to the lack of domain interactions stabilizing the intact protein.Unfolding/folding kinetics of the 10-kDa fragment coincide with the second phase of the bimodal transition of intact yII-crystallin, in agreement with independent sequential folding and modular assembly of the domains within the native molecule. Domains, as distinct structural regions within globular proteins, represent autonomous cooperative folding units. As taken from their occurrence in all large proteins, 'modular assembly' [l] is a general strategy of protein folding offering long polypeptide chains the advantage of rapid self-organization. The discovery of coding and non-coding sequences in genomic DNA, and the correlation of individual protein domains with separate exons suggests 'genes-in-pieces' to imply 'proteins-in-pieces' as a common feature in the processes of transcription, splicing and translation. In certain cases, domains have been shown to originate from gene duplications. In cases where active sites reside on different domains or at domain interfaces, domain interactions may have functional significance, e.g. in terms of hinge movements, or mutual stabilization.yII-Crystallin is a typcial two-domain protein.As taken from high-resolution X-ray analysis, the N-and C-terminal halves of the molecule show twofold symmetry, each of the domains representing a sandwich consisting of two fourstrand pleated sheets organized in 'Greek key' motifs [2]. Both sequence homology and molecular topology suggest that 711-crystallin is the product of gene duplication. The high symmetry and the distinct charge pattern on the surface of the molecule may be responsible for the anomalously high stability of the molecule [3]. Whether, in this context, the domains are intrinsically stable, or whether domain interactions conCorrespondence to R. Jaenicke,
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.