5
views
0
recommends
+1 Recommend
1 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Wet-Etched Microchamber Array Digital PCR Chip for SARS-CoV-2 Virus and Ultra-Early Stage Lung Cancer Quantitative Detection

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          We report a novel design of chamber-based digital polymerase chain reaction (cdPCR) chip structure. Using a wet etching process and silicon-glass bonding, the chamber size can be adjusted independently of the process and more feasibly in a normal lab. In addition, the structure of the chip is optimized through hydrodynamic computer simulations to eliminate dead space when the sample is injected into the chip. The samples will be distributed to each separated microchambers for an isolated reaction based on Poisson distribution. Due to the difference in expansion coefficients, isolation of the sample in the microchambers by the oil phase on top ensures homogeneity and independence of the sample in the microchambers. The prepared microarray cdPCR chip enables high-throughput and high-sensitivity quantitative measurement of the SARS-CoV-2 virus gene and the mutant lung cancer gene. We applied the chip for the detection of different concentrations of the mix containing the open reading frame 1ab (ORF1ab) gene, the most specific and conservative gene region of the SARS-CoV-2 virus. In addition to this, we also successfully detected the fluorescence of the epidermal growth factor receptor (EGFR) mutant gene in independent microchambers. At a throughput of 46 200 microchambers, solution mixtures containing both genes were successfully tested quantitatively, with a detection limit of 10 copies/μL. Importantly, the chips are individually inexpensive and easy to industrialize. In addition, the microarray can provide a unified solution for other viral sequences, cancer marker assay development, and point-of-care testing (POCT).

          Related collections

          Most cited references29

          • Record: found
          • Abstract: found
          • Article: found
          Is Open Access

          SARS-CoV-2 within-host diversity and transmission

          A year into the severe acute respiratory syndrome coronavirus 2 pandemic, we are experiencing waves of new variants emerging. Some of these variants have worrying functional implications, such as increased transmissibility or antibody treatment escape. Lythgoe et al. have undertaken in-depth sequencing of more than 1000 hospital patients' isolates to find out how the virus is mutating within individuals. Overall, there seem to be consistent and reproducible patterns of within-host virus diversity. The authors observed only one or two variants in most samples, but a few carried many variants. Although the evidence indicates strong purifying selection, including in the spike protein responsible for viral entry, the authors also saw evidence for transmission clusters associated with households and other possible superspreader events. After transmission, most variants fizzled out, but occasionally some initiated ongoing transmission and wider dissemination. Science , this issue p. eabg0821 More than a thousand deep-sequenced clinical samples from the UK reveal that SARS-CoV-2 has limited genetic diversity within most individuals. INTRODUCTION Genome sequencing at an unprecedented scale during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is helping to track spread of the virus and to identify new variants. Most of this work considers a single consensus sequence for each infected person. Here, we looked beneath the consensus to analyze genetic variation within viral populations making up an infection and studied the fate of within-host mutations when an infection is transmitted to a new individual. Within - host diversity offers the means to help confirm direct transmission and identify new variants of concern. RATIONALE We sequenced 1313 SARS-CoV-2 samples from the first wave of infection in the United Kingdom. We characterized within-host diversity and dynamics in the context of transmission and ongoing viral evolution. RESULTS Within-host diversity can be described by the number of intrahost single nucleotide variants (iSNVs) occurring above a given minor allele frequency (MAF) threshold. We found that in lower-viral-load samples, stochastic sampling effects resulted in a higher variance in MAFs, leading to more iSNVs being detected at any threshold. Based on a subset of 27 pairs of high-viral-load replicate RNA samples (>50,000 uniquely mapped veSEQ reads, corresponding to a cycle threshold of ~22), iSNVs with a minimum 3% MAF were highly reproducible. Comparing samples from two time points from 41 individuals, taken on average 6 days apart (interquartile ratio 2 to 10), we observed a dynamic process of iSNV generation and loss. Comparing iSNVs among 14 household contact pairs, we estimated transmission bottleneck sizes of one to eight viruses. Consensus differences between individuals in the same household, where sample depth allowed iSNV detection, were explained by the presence of an iSNV at the same site in the paired individual, consistent with direct transmission leading to fixation. We next focused on a set of 563 high-confidence iSNV sites that were variant in at least one high-viral-load sample (>50,000 uniquely mapped); low-confidence iSNVs unlikely to represent genomic diversity were excluded. Within-host diversity was limited in high-viral-load samples (mean 1.4 iSNVs per sample). Two exceptions, each with >14 iSNVs, showed variant frequencies consistent with coinfection or contamination. Overall, we estimated that 1 to 2% of samples in our dataset were coinfected and/or contaminated. Additionally, one sample was coinfected with another coronavirus (OC43), with no detectable impact on diversity. The ratio of nonsynonymous to synonymous ( dN/dS ) iSNVs was consistent with within-host purifying selection when estimated across the whole genome [ dN/dS = 0.55, 95% confidence interval (95% CI) = 0.49 to 0.61] and for the Spike gene ( dN/dS = 0.60, 95% CI = 0.45 to 0.82). Nevertheless, we observed Spike variants in multiple samples that have been shown to increase viral infectivity (L5F) or resistance to antibodies (G446V and A879V). We observed a strong association between high-confidence iSNVs and a consensus change on the phylogeny (153 cases), consistent with fixation after transmission or de novo mutations reaching consensus. Shared variants that never reached consensus (261 cases) were not phylogenetically associated. CONCLUSION Using robust methods to call within-host variants, we uncovered a consistent pattern of low within-host diversity, purifying selection, and narrow transmission bottlenecks. Within-host emergence of vaccine and therapeutic escape mutations is likely to be relatively rare, at least during early infection, when viral loads are high, but the observation of immune-escape variants in high-viral-load samples underlines the need for continued vigilance. Diagram showing low SARS-CoV-2 within-host genetic diversity and narrow transmission bottleneck. Individuals with high viral load typically have few, if any, within-host variants. Narrow transmission bottlenecks mean that the major variant in the source individual was typically transmitted and the minor variants lost. Occasionally, the minor variant was transmitted, leading to a consensus change, or multiple variants were transmitted, resulting in a mixed infection. Credit: FontAwesome, licensed under CC BY 4.0. Extensive global sampling and sequencing of the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have enabled researchers to monitor its spread and to identify concerning new variants. Two important determinants of variant spread are how frequently they arise within individuals and how likely they are to be transmitted. To characterize within-host diversity and transmission, we deep-sequenced 1313 clinical samples from the United Kingdom. SARS-CoV-2 infections are characterized by low levels of within-host diversity when viral loads are high and by a narrow bottleneck at transmission. Most variants are either lost or occasionally fixed at the point of transmission, with minimal persistence of shared diversity, patterns that are readily observable on the phylogenetic tree. Our results suggest that transmission-enhancing and/or immune-escape SARS-CoV-2 variants are likely to arise infrequently but could spread rapidly if successfully transmitted.
            Bookmark
            • Record: found
            • Abstract: not found
            • Article: not found

            Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.

              Bookmark
              • Record: found
              • Abstract: found
              • Article: found

              Simultaneous dual‐gene diagnosis of SARS‐CoV‐2 based on CRISPR/Cas9‐mediated lateral flow assay

              Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.
                Bookmark

                Author and article information

                Journal
                ACS Omega
                ACS Omega
                ao
                acsodf
                ACS Omega
                American Chemical Society
                2470-1343
                07 January 2022
                18 January 2022
                : 7
                : 2
                : 1819-1826
                Affiliations
                []State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences , Shanghai 200050, China
                []Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences , Beijing 100049, China
                [§ ]School of Life Sciences, Shanghai Normal University , Shanghai 200235, China
                []Faculty of Electrical Engineering and Computer Science, Ningbo University , Ningbo 315211, China
                Author notes
                Author information
                https://orcid.org/0000-0002-2560-2417
                https://orcid.org/0000-0002-1255-2764
                Article
                10.1021/acsomega.1c05082
                8751011
                35036821
                7b6bae68-72b0-4897-b871-a6cda90dae93
                © 2022 The Authors. Published by American Chemical Society

                This article is made available via the PMC Open Access Subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 14 September 2021
                : 22 December 2021
                Funding
                Funded by: National Natural Science Foundation of China, doi 10.13039/501100001809;
                Award ID: 61804158
                Funded by: Shanghai Rising-Star Program, doi 10.13039/501100013105;
                Award ID: 21QC1400700
                Funded by: Shanghai Municipal Education Commission, doi 10.13039/501100003395;
                Award ID: 21PJ1415000
                Funded by: Ministry of Science and Technology of the People''s Republic of China, doi 10.13039/501100002855;
                Award ID: 2021YFB3201402
                Funded by: Chinese Academy of Sciences, doi 10.13039/501100002367;
                Award ID: GJJSTD20210006
                Categories
                Article
                Custom metadata
                ao1c05082
                ao1c05082

                Comments

                Comment on this article