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1.
Clin Microbiol Rev ; : e0016821, 2022 Mar 08.
Article in English | MEDLINE | ID: covidwho-1731254

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples. In contrast, digital PCR (dPCR), the third generation of PCR, has been shown to be more effective than the gold standard, RT-qPCR, in detecting low viral loads in samples. In this review article, we selected publications to show the broad-spectrum applications of dPCR, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2, while comparing it analytically to the gold standard, RT-qPCR. In summary, it is evident that the specificity, sensitivity, reproducibility, and detection limits of RT-dPCR are generally unaffected by common factors that may affect RT-qPCR. As this is the first time that dPCR is being tested in an outbreak of such a magnitude, knowledge of its applications will help chart a course for future diagnosis and monitoring of infectious disease outbreaks.

2.
Anal Chem ; 94(10): 4522-4530, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1721381

ABSTRACT

Positive controls made of viral gene components are essential to validate the performance of diagnostic assays for pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, most of them are target-specific, limiting their application spectrum when validating assays beyond their specified targets. The use of an inactivated whole-virus RNA reference standard could be ideal, but RNA is a labile molecule that needs cold chain storage and transportation to preserve its integrity and activity. The cold chain process stretches the already dwindling storage capacities, incurs huge costs, and limits the distribution of reference materials to low-resource settings. To circumvent these issues, we developed an inactivated whole-virus SARS-CoV-2 RNA reference standard and studied its stability in silk fibroin matrices, i.e., silk solution (SS) and silk film (SF). Compared to preservation in nuclease-free water (ddH2O) and SS, SF was more stable and could preserve the SARS-CoV-2 RNA reference standard at room temperature for over 21 weeks (∼6 months) as determined by reverse transcription polymerase chain reaction (RT-PCR). The preserved RNA reference standard in SF was able to assess the limits of detection of four commercial SARS-CoV-2 RT-PCR assays. In addition, SF is compatible with RT-PCR reactions and can be used to preserve a reaction-ready primer and probe mix for RT-PCR at ambient temperatures without affecting their activity. Taken together, these results offer extensive flexibility and a simpler mechanism of preserving RNA reference materials for a long time at ambient temperatures of ≥25 °C, with the possibility of eliminating cold chains during storage and transportation.


Subject(s)
COVID-19 , RNA, Viral , COVID-19/diagnosis , Humans , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Silk
4.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-318163

ABSTRACT

Outbreaks of coronavirus disease 2019 (COVID-19) have been recorded in different countries across the globe. The virus is highly contagious, hence early detection, isolation, and quarantine of infected patients will play an important role in containing the viral spread. Diagnosis in a mobile lab can aid to find infected patients in time. Here, we develop a field-deployable diagnostic workflow that can reliably detect COVID-19. Instruments used in this workflow could easily fit in a mobile cabin hospital and also be installed in the community. Different steps from sample inactivation to detection were optimized to find the fastest steps and portable instruments in detection of COVID-19. Each step was compared to that of the normal laboratory diagnosis set-up. From the results, our proposed workflow (80 min) was two times faster compared to that of the normal laboratory workflow (183 min) and a maximum of 32 samples could be detected at each run. Additionally, we showed that using 1% Rewocid WK-30 could inactivate the novel coronavirus directly without affecting the overall detection results. Comparison of our workflow using an in-house assay to that of a commercially acquired assay produced highly reliable results. From the 250 hospital samples tested, there was a high concordance 247/250 (98.8%) between the two assays. The in-house assay sensitivity and specificity were 116/116 (100%) and 131/134 (97.8%) compared to that of the commercial assay. Based on these results, we believe that our workflow is fast, reliable, adaptable and most importantly, field deployable.

5.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-317641

ABSTRACT

SARS-CoV-2, the causative agent of coronavirus disease 19 (COVID 19), is responsible for the ongoing pandemic but still lacks approved antivirals. Repurposing pre-existing FDA approved drugs presents a rapid approach for new therapeutic options. In the present study, we report that three pre-existing FDA-approved drugs, i.e., vapreotide, grazoprevir, and simeprevir, inhibit the replication of SARS-CoV-2 in cells. The E50 values of vapreotide, grazoprevir, and simeprevir against SARS-CoV-2 in Vero E6 cells was 3.98 ± 0.35 μM, 2.08 ± 0.13 μM, and 1.41 ± 0.12 μM, respectively. In vitro biochemical experiments further revealed that vapreotide, grazoprevir, and simeprevir efficiently inhibits the unwinding activity of the Nsp13 helicase of SARS-CoV-2 with IC50 values of ⁓10, ⁓2.5, and ⁓1.25 µM, respectively, providing signs for understanding their antiviral mechanism of action. Given their good safety profiles in their original indications, our study offices new insights in repurposing these drugs alone or in combination with other antivirals in the global fighting against SARS-CoV-2.Funding: This work was financially supported by the Youth Innovation Promotion Association CAS (to H.Y.), and the National Natural Science Foundation of China (No. 31770192 and No. 32070187 to H.Y.).Conflict of Interest: The authors declare no competing interests.

7.
Anal Chim Acta ; 1200: 339590, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1682826

ABSTRACT

The global public health crisis and economic losses resulting from the current novel coronavirus disease (COVID-19) pandemic have been dire. The most used real-time reverse transcription polymerase chain reaction (RT-PCR) method needs expensive equipment, technical expertise, and a long turnaround time. Therefore, there is a need for a rapid, accurate, and alternative technique of diagnosis that is deployable at resource-poor settings like point-of-care. This study combines heat deactivation and a novel mechanical lysis method by bead beating for quick and simple sample preparation. Then, using an optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to target genes encoding the open reading frame 8 (ORF8), spike and nucleocapsid proteins of the novel coronavirus, SARS-CoV-2. The test results can be read simultaneously in fluorometric and colorimetric readouts within 40 min from sample collection. We also calibrated a template transfer tool to simplify sample addition into LAMP reactions when pipetting skills are needed. Most importantly, validation of the direct RT-LAMP system based on multiplexing primers S1:ORF8 in a ratio (1:0.8) using 143 patients' nasopharyngeal swab samples showed a diagnostic performance of 99.30% accuracy, with 98.81% sensitivity and 100% selectivity, compared to commercial RT-PCR kits. Since our workflow does not rely on RNA extraction and purification, the time-to-result is two times faster than other workflows with FDA emergency use authorization. Considering all its strengths: speed, simplicity, accuracy and extraction-free, the system can be useful for optimal point-of-care testing of COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2/genetics , Sensitivity and Specificity
8.
Viruses ; 14(2)2022 02 07.
Article in English | MEDLINE | ID: covidwho-1674827

ABSTRACT

A new SARS-CoV-2 variant B.1.1.529 was named by the WHO as Omicron and classified as a Variant of Concern (VOC) on 26 November 2021. Because this variant has more than 50 mutations, including 30 mutations on the spike, it has generated a lot of concerns on the potential impacts of the VOC on COVID-19. Here through ELISA assays using the recombinant RBD proteins with sequences the same to that of SARS-CoV-2 WIV04 (lineage B.1), the Delta variant and the Omicron variant as the coating antigens, the binding capabilities between the RBDs and the antibodies in COVID-19 convalescent sera and vaccine sera after two doses of the inactivated vaccine produced by Sinopharm WIBP are compared with each other. The results showed that the Omicron variant may evade antibodies induced by the ancestral strain and by the inactivated vaccine, with significant reduction in the binding capability of its RBD much greater than that of the Delta variant.


Subject(s)
Antibodies, Viral/metabolism , Binding Sites, Antibody/physiology , COVID-19 Vaccines/immunology , COVID-19/immunology , Convalescence , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/blood , Antibodies, Viral/immunology , Humans , Immune Evasion , Mutation , Neutralization Tests , Vaccines, Inactivated/immunology
9.
Microbiol Spectr ; 10(1): e0143821, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1608700

ABSTRACT

With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/µL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.


Subject(s)
COVID-19/virology , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , Genome, Viral , High-Throughput Nucleotide Sequencing , Mutation , Nucleic Acid Amplification Techniques/trends , Pharynx/virology , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/classification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/classification , Spike Glycoprotein, Coronavirus/genetics
10.
Biosensors (Basel) ; 12(1)2021 Dec 26.
Article in English | MEDLINE | ID: covidwho-1581023

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.


Subject(s)
COVID-19 Testing , COVID-19 , CRISPR-Cas Systems , COVID-19/diagnosis , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral , SARS-CoV-2 , Sensitivity and Specificity
11.
Int J Antimicrob Agents ; 59(1): 106499, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1587677

ABSTRACT

In a bid to contain the current COVID-19 (coronavirus disease 2019) pandemic, various countermeasures have been applied. To date, however, there is a lack of an effective drug for the treatment of COVID-19. Through molecular modelling studies, simeprevir, a protease inhibitor approved for the management of hepatitis C virus infection, has been predicted as a potential antiviral against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative agent of COVID-19. Here we assessed the efficacy of simeprevir against SARS-CoV-2 both in vitro in Vero E6 cells and in vivo in a human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model. The results showed that simeprevir could inhibit SARS-CoV-2 replication in Vero E6 cells with a half-maximal effective concentration (EC50) of 1.41 ± 0.12 µM. In a transgenic hACE2 mouse model of SARS-CoV-2 infection, intraperitoneal administration of simeprevir at 10 mg/kg/day for 3 consecutive days failed to suppress viral replication. These findings collectively imply that simeprevir does not inhibit SARS-CoV-2 in vivo and therefore do not support its application as a treatment against COVID-19 at a dosage of 10 mg/kg/day.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Simeprevir/pharmacology , Virus Replication/drug effects , Animals , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Chlorocebus aethiops , Dose-Response Relationship, Drug , Humans , Male , Mice , Mice, Transgenic , Negative Results , Protease Inhibitors/therapeutic use , Simeprevir/therapeutic use , Vero Cells
12.
J Adv Res ; 36: 201-210, 2022 02.
Article in English | MEDLINE | ID: covidwho-1536631

ABSTRACT

Introduction: The COVID-19 global epidemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a great public health emergency. Discovering antiviral drug candidates is urgent for the prevention and treatment of COVID-19. Objectives: This work aims to discover natural SARS-CoV-2 inhibitors from the traditional Chinese herbal medicine licorice. Methods: We screened 125 small molecules from Glycyrrhiza uralensis Fisch. (licorice, Gan-Cao) by virtual ligand screening targeting the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. Potential hit compounds were further evaluated by ELISA, SPR, luciferase assay, antiviral assay and pharmacokinetic study. Results: The triterpenoids licorice-saponin A3 (A3) and glycyrrhetinic acid (GA) could potently inhibit SARS-CoV-2 infection, with EC50 of 75 nM and 3.17 µM, respectively. Moreover, we reveal that A3 mainly targets the nsp7 protein, and GA binds to the spike protein RBD of SARS-CoV-2. Conclusion: In this work, we found GA and A3 from licorice potently inhibit SARS-CoV-2 infection by affecting entry and replication of the virus. Our findings indicate that these triterpenoids may contribute to the clinical efficacy of licorice for COVID-19 and could be promising candidates for antiviral drug development.


Subject(s)
COVID-19 , Glycyrrhiza , Triterpenes , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Triterpenes/pharmacology
14.
J Biosaf Biosecur ; 2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1506533

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease 2019 (COVID-19) pandemic has crippled several countries across the globe posing a serious global public health challenge. Despite the massive rollout of vaccines, molecular diagnosis remains the most important method for timely isolation, diagnosis, and control of COVID-19. Several molecular diagnostic tools have been developed since the beginning of the pandemic with some even gaining emergency use authorization (EUA) from the United States (US) Food and Drug Administration (FDA) for in vitro diagnosis of SARS-CoV-2. Herein, we discuss the working principles of some commonly used molecular diagnostic tools for SARS-CoV-2 including nucleic acid amplification tests (NAATs), isothermal amplification tests (IATs), and rapid diagnostic tests (RDTs). To ensure successful detection while minimizing the risk of cross-infection and misdiagnosis when using these diagnostic tools, laboratories should adhere to proper biosafety practices. Hence, we also present the common biosafety practices that may ensure the successful detection of SARS-CoV-2 from specimens while protecting laboratory workers and non-suspecting individuals from being infected. From this review article, it is clear that the SARS-CoV-2 pandemic has led to an increase in molecular diagnostic tools and the formation of new biosafety protocols that may be important for future and ongoing outbreaks.

15.
Cell Discov ; 7(1): 67, 2021 Aug 17.
Article in English | MEDLINE | ID: covidwho-1360193

ABSTRACT

One of the best ways to control COVID-19 is vaccination. Among the various SARS-CoV-2 vaccines, inactivated virus vaccines have been widely applied in China and many other countries. To understand the underlying protective mechanism of these vaccines, it is necessary to systematically analyze the humoral responses that are triggered. By utilizing a SARS-CoV-2 microarray with 21 proteins and 197 peptides that fully cover the spike protein, antibody response profiles of 59 serum samples collected from 32 volunteers immunized with the inactivated virus vaccine BBIBP-CorV were generated. For this set of samples, the microarray results correlated with the neutralization titers of the authentic virus, and two peptides (S1-5 and S2-22) were identified as potential biomarkers for assessing the effectiveness of vaccination. Moreover, by comparing immunized volunteers to convalescent and hospitalized COVID-19 patients, the N protein, NSP7, and S2-78 were identified as potential biomarkers for differentiating COVID-19 patients from individuals vaccinated with the inactivated SARS-CoV-2 vaccine. The comprehensive profile of humoral responses against the inactivated SARS-CoV-2 vaccine will facilitate a deeper understanding of the vaccine and provide potential biomarkers for inactivated virus vaccine-related applications.

16.
Sci Total Environ ; 797: 149085, 2021 Nov 25.
Article in English | MEDLINE | ID: covidwho-1313422

ABSTRACT

The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Azides , Humans , Pandemics , Propidium/analogs & derivatives , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
17.
Virol Sin ; 36(5): 924-933, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1225063

ABSTRACT

As a respiratory tract virus, SARS-CoV-2 infected people through contacting with the upper respiratory tract first. Previous studies indicated that microbiota could modulate immune response against pathogen infection. In the present study, we performed metagenomic sequencing of pharyngeal swabs from eleven patients with COVID-19 and eleven Non-COVID-19 patients who had similar symptoms such as fever and cough. Through metagenomic analysis of the above two groups and a healthy group from the public data, there are 6502 species identified in the samples. Specifically, the Pielou index indicated a lower evenness of the microbiota in the COVID-19 group than that in the Non-COVID-19 group. Combined with the linear discriminant analysis (LDA) and the generalized linear model, eighty-one bacterial species were found with increased abundance in the COVID-19 group, where 51 species were enriched more than 8 folds. The top three enriched genera were Streptococcus, Prevotella and Campylobacter containing some opportunistic pathogens. More interestingly, through experiments, we found that two Streptococcus strains, S. suis and S. agalactiae, could stimulate the expression of ACE2 of Vero cells in vitro, which may promote SARS-CoV-2 infection. Therefore, these enriched pathogens in the pharynxes of COVID-19 patients may involve in the virus-host interactions to affect SARS-CoV-2 infection and cause potential secondary bacterial infections through changing the expression of the viral receptor ACE2 and/or modulate the host's immune system.


Subject(s)
COVID-19 , Microbiota , Animals , Chlorocebus aethiops , Humans , Metagenomics , SARS-CoV-2 , Vero Cells
18.
J Vis Exp ; (169)2021 03 31.
Article in English | MEDLINE | ID: covidwho-1192273

ABSTRACT

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , DNA Primers , Humans , Pandemics , RNA, Viral/genetics , Reverse Transcription , Sensitivity and Specificity
19.
Virol Sin ; 36(5): 901-912, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1182321

ABSTRACT

Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing. While the rapid acquisition of SARS-CoV-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background. To address this issue, we modified and evaluated an approach by utilizing SARS-CoV-2-specific amplicon amplification and Oxford Nanopore PromethION platform. This workflow started with the throat swab of the COVID-19 patient, combined reverse transcript PCR, and multi-amplification in one-step to shorten the experiment time, then can quickly and steadily obtain high-quality SARS-CoV-2 genome within 24 h. A comprehensive evaluation of the method was conducted in 42 samples: the sequencing quality of the method was correlated well with the viral load of the samples; high-quality SARS-CoV-2 genome could be obtained stably in the samples with Ct value up to 39.14; data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20; variation analysis indicated that the method can detect the existing and emerging genomic mutations as well; Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing, making it as low as 0.4/10,000 bp. In summary, high-quality SARS-CoV-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARS-CoV-2.


Subject(s)
COVID-19 , Nanopore Sequencing , Genome, Viral , High-Throughput Nucleotide Sequencing , Humans , Pandemics , RNA, Viral/genetics , SARS-CoV-2
20.
Cell Discov ; 7(1): 19, 2021 Mar 30.
Article in English | MEDLINE | ID: covidwho-1160081

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a recently developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive, and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance, and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.

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