scholarly journals Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR

Diagnostics ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1241
Author(s):  
Agathe Boudet ◽  
Robin Stephan ◽  
Sophie Bravo ◽  
Milène Sasso ◽  
Jean-Philippe Lavigne
Keyword(s):  
Next Generation Sequencing ◽  
Virus Evolution ◽  
Screening Strategy ◽  
Next Generation ◽  
Rt Pcr ◽  
Screening Assay ◽  
Pcr Screening ◽  
S Gene ◽  
The Uk ◽  
Generation Sequencing

Since January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution.

scholarly journals Next-generation sequencing and RT-PCR to identify a 32-day SARS-CoV-2 carrier

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Lingjie Song ◽  
Guibao Xiao ◽  
Ruixiang Tang ◽  
Xianqin Zhang ◽  
Zhan Gao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Rt Pcr ◽  
Generation Sequencing

Discovery of mutations in Chenopodium quinoa Willd through EMS mutagenesis and mutation screening using pre-selection phenotypic data and next-generation sequencing

2018 ◽  
Vol 156 (10) ◽  
pp. 1196-1204 ◽  
Author(s):  
Camilo Mestanza ◽  
Ricardo Riegel ◽  
Santiago C. Vásquez ◽  
Diana Veliz ◽  
Nicolás Cruz-Rosero ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mutation Screening ◽  
Chenopodium Quinoa ◽  
Acetolactate Synthase ◽  
Screening Strategy ◽  
Next Generation ◽  
Induced Mutations ◽  
Phenotypic Data ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

AbstractQuinoa (Chenopodium quinoaWilld) is a dicotyledonous annual species belonging to the family Amaranthaceae, which is nutritionally well balanced in terms of its oil, protein and carbohydrate content. Targeting-induced local lesions in genomes (the TILLING strategy) was employed to find mutations in acetolactate synthase (AHAS) genes in a mutant quinoa population. TheAHASgenes were targeted because they are common enzyme target sites for five herbicide groups. Ethyl methane sulfonate (EMS) was used to induce mutations in theAHASgenes; it was found that 2% EMS allowed a mutation frequency of one mutation every 203 kilobases to be established. In the mutant population created, a screening strategy using pre-selection phenotypic data and next-generation sequencing (NGS) allowed identification of a mutation that alters the amino acid composition of this species (nucleotide 1231 codon GTT→ATT, Val→Ile); however, this mutation did not result in herbicide resistance. The current work shows that TILLING combined with the high-throughput of NGS technologies and an overlapping pool design provides an efficient and economical method for detecting induced mutations in pools of individuals.

scholarly journals SARS-CoV-2 as a Trigger in the Development of Tourette’s-Like Symptoms: A Case Report

2021 ◽  
Author(s):  
Sabine Hazan ◽  
Sheldon Jordan
Keyword(s):  
Case Report ◽  
Next Generation Sequencing ◽  
Gut Microbiota ◽  
Dramatic Improvement ◽  
Next Generation ◽  
Rt Pcr ◽  
Gastrointestinal Microbiota ◽  
Next Generation Sequencing Ngs ◽  
The Impact ◽  
Generation Sequencing

Abstract Background: Reports have been surfacing surrounding CNS-associated symptoms in individuals affected by coronavirus disease 19 (COVID-19). Tourette syndrome is a neuropsychiatric disorder with usual onset in childhood. Gut microbiota can affect central physiology and function via the microbiota-gut-brain axis. The authors of this case report describe Tourette’s-like symptoms in a patient resulting from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection disrupting gut microbiota. Case Presentation: This case involves a 16-year-old female that developed acute onset Tourette’s-like symptoms along with neuropsychiatric symptoms after exposure to and infection from SARS-CoV-2. The patient had negative nasopharyngeal (NP) real-time reverse transcription-PCR (RT-PCR) tests for SARS-CoV-2 on five occasions from August of 2020 through June of 2021. The patient’s symptoms continued to worsen over the next six months until next-generation sequencing (NGS) revealed SARS-CoV-2 in her stool. Her treatment was adjusted as NGS revealed SARS-CoV-2 in her stool. Repair of the gastrointestinal microbiota, treatment with nutraceutical and pharmaceutical agents, as well as alterations in her surroundings resulted in dramatic improvement in the microbiome and a significant reduction of symptoms.Discussion: The use of (RT-PCR) testing to determine the presence or absence of SARS-CoV-2 may be inadequate and inaccurate for individuals that have been exposed to the virus. In addition, the impact of SARS-CoV-2 infection of the GI tract may cause significant havoc in the gut microbiota. Additional testing, eradication of infectious agents, as well as restoration of the gut microbiome are needed to effectively manage and treat this condition. The patient’s symptoms worsened over the next six months until next-generation sequencing (NGS) revealed SARS-CoV-2 in her stool and her treatment was adjusted. Treatment with nutraceuticals and alterations in her surroundings was followed by a more normal microbiome and a dramatic reduction in symptoms.

scholarly journals Next-Generation Sequencing-Based Detection of Common Bean Viruses in Wild Plants from Tanzania and Their Mechanical Transmission to Common Bean Plants

Plant Disease ◽  
2021 ◽  
Author(s):  
Beatrice Mwaipopo ◽  
Minna-Liisa Rajamäki ◽  
Neema Ngowi ◽  
Susan N’chimbi Msolla ◽  
P Njau ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Common Bean ◽  
Mosaic Virus ◽  
Viral Disease ◽  
Wild Plants ◽  
Next Generation ◽  
Rt Pcr ◽  
Bean Plants ◽  
Generation Sequencing ◽  
Common Bean Plants

Viral diseases are a major threat for common bean production. In recent surveys, >15 different viruses belonging to 11 genera were shown to infect common bean (Phaseolus vulgaris L.) in Tanzania. Management of viruses requires an understanding of how they survive from one season to the next. In this study, we explored the possibility that alternative host plants have a central role in the survival of common bean viruses. We used next-generation sequencing (NGS) techniques to sequence virus-derived small interfering RNAs, together with conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect viruses in wild plants. Leaf samples for RNA extraction and NGS were collected from 1,430 wild plants around and within common bean fields in four agricultural zones in Tanzania. At least partial genome sequences of viruses potentially belonging to 25 genera were detected. The greatest virus diversity was detected in the eastern and northern zones, whereas wild plants in the Lake zone and especially in the southern highlands zone showed only a few viruses. RT-PCR analysis of all the collected plant samples confirmed the presence of yam bean mosaic virus and peanut mottle virus in wild legume plants. Of all viruses detected, only two viruses, cucumber mosaic virus and a novel bromovirus related to cowpea chlorotic mottle virus and brome mosaic virus, were mechanically transmitted from wild plants to common bean plants. The data generated in this study are crucial for development of viral disease management strategies and predicting crop viral disease outbreaks in different agricultural regions in Tanzania and beyond.

scholarly journals Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

2020 ◽  
Author(s):  
Chenyu Li ◽  
David N. Debruyne ◽  
Julia Spencer ◽  
Vidushi Kapoor ◽  
Lily Y. Liu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Multiplex Pcr ◽  
High Sensitivity ◽  
Detection Methods ◽  
Next Generation ◽  
Rt Pcr ◽  
Copy Numbers ◽  
Virus Rna ◽  
Highly Sensitive ◽  
Generation Sequencing

AbstractMany detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

scholarly journals Chronic SARS-CoV-2 infection and viral evolution in a hypogammaglobulinaemic individual.

2021 ◽  
Author(s):  
Maia Kavanagh Williamson ◽  
Fergus Hamilton ◽  
Stephanie Hutchings ◽  
Hannah M Pymont ◽  
Mark Hackett ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Amino Acid ◽  
Selective Pressure ◽  
Viral Evolution ◽  
Viral Escape ◽  
Spike Protein ◽  
Next Generation ◽  
Rt Pcr ◽  
The Face ◽  
Generation Sequencing

There is widespread interest in the capacity for SARS-CoV-2 evolution in the face of selective pressures from host immunity, either naturally acquired post-exposure or from vaccine acquired immunity. Allied to this is the potential for long perm persistent infections within immune compromised individuals to allow a broader range of viral evolution in the face of sub-optimal immune driven selective pressure. Here we report on an immunocompromised individual who is hypogammaglobulinaemic and was persistently infected with SARS-CoV-2 for over 290 days, the longest persistent infection recorded in the literature to date. During this time, nine samples of viral nucleic acid were obtained and analysed by next-generation sequencing. Initially only a single mutation (L179I) was detected in the spike protein relative to the prototypic SARS-CoV-2 Wuhan-Hu-1 isolate, with no further changes identified at day 58. However, by day 155 the spike protein had acquired a further four amino acid changes, namely S255F, S477N, H655Y and D1620A and a two amino acid deletion (ΔH69/ΔV70). Infectious virus was cultured from a nasopharyngeal sample taken on day 155 and next-generation sequencing confirmed that the mutations in the virus mirrored those identified by sequencing of the corresponding swab sample. The isolated virus was susceptible to remdesivir in vitro, however a 17-day course of remdesivir started on day 213 had no effect on the viral RT-PCR cycle threshold (Ct) value. On day 265 the patient was treated with the combination of casirivimab and imdevimab. The patient experienced progressive resolution of all symptoms over the next 8 weeks and by day 311 the virus was no longer detectable by RT-PCR. The ΔH69/ΔV70 deletion in the N-terminus of the spike protein which arose in our patient is also present in the B.1.1.7 variant of concern and has been associated with viral escape mutagenesis after treatment of another immunocompromised patient with convalescent plasma. Our data confirms the significance of this deletion in immunocompromised patients but illustrates it can arise independently of passive antibody transfer, suggesting the deletion may be an enabling mutation that compensates for distant changes in the spike protein that arise under selective pressure.

scholarly journals A pandemic in the age of next-generation sequencing

2021 ◽  
Author(s):  
Angela H. Beckett ◽  
Kate F. Cook ◽  
Samuel C. Robson
Keyword(s):  
Public Health ◽  
Next Generation Sequencing ◽  
Infection Control ◽  
Control Measures ◽  
Molecular Medicine ◽  
Next Generation ◽  
Healthcare Settings ◽  
The Uk ◽  
The Impact ◽  
Generation Sequencing

Since December 2019, the world has found itself rocked by the emergence of a highly contagious novel coronavirus disease, COVID-19, caused by the virus SARS-CoV-2. The global scientific community has rapidly come together to understand the virus and identify potential treatments and vaccine strategies to minimise the impact on public health. Key to this has been the use of cutting-edge technological advances in DNA and RNA sequencing, allowing identification of changes in the viral genome sequence as the infection spreads. This approach has allowed a widespread ‘genomic epidemiology’ approach to infection control, whereby viral transmission (e.g. in healthcare settings) can be detected not only by epidemiological assessment, but also by identifying similarities between viral sub-types among individuals. The UK has been at the forefront of this response, with researchers collaborating with public health agencies and NHS Trusts across the UK to form the COVID-19 Genomics UK (COG-UK) Consortium. Genomic surveillance at this scale has provided critical insight into the virulence and transmission of the virus, enabling near real-time monitoring of variants of concern and informing infection control measures on local, national and global scales. In the future, next-generation sequencing technologies, such as nanopore sequencing, are likely to become ubiquitous in diagnostic and healthcare settings, marking the transition to a new era of molecular medicine.

scholarly journals Validation of Saliva and Self-Administered Nasal Swabs for COVID-19 Testing

2020 ◽  
Author(s):  
Alvin Kuo Jing Teo ◽  
Yukti Choudhury ◽  
Iain Beehuat Tan ◽  
Chae Yin Cher ◽  
Shi Hao Chew ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Migrant Workers ◽  
Phylogenetic Analyses ◽  
Kappa Statistic ◽  
Sample Type ◽  
Next Generation ◽  
Rt Pcr ◽  
Male Adult ◽  
Public Health Decision ◽  
Generation Sequencing

Background Active cases of COVID-19 has primarily been diagnosed via RT-PCR of nasopharyngeal (NP) swabs. Saliva and self-administered nasal (SN) swabs can be collected safely without trained staff. We aimed to test the sensitivity of naso-oropharyngeal saliva and SN swabs compared to NP swabs in a large cohort of migrant workers in Singapore. Methods We recruited 200 male adult subjects: 45 with acute respiratory infection, 104 asymptomatic close contacts, and 51 confirmed COVID-19 cases. Each subject underwent NP swab, SN swab and saliva collection for RT-PCR testing at 1 to 3 timepoints. We additionally used a direct-from-sample amplicon-based next-generation sequencing (NGS) workflow to establish phylogeny. Results Of 200 subjects, 91 and 46 completed second and third rounds of testing, respectively. Of 337 sets of tests, there were 150 (44.5%) positive NP swabs, 127 (37.7%) positive SN swabs, and 209 (62.0%) positive saliva. Test concordance between different sample sites was good, with a kappa statistic of 0.616 for NP and SN swabs, and 0.537 for NP and saliva. In confirmed symptomatic COVID-19 subjects, the likelihood of a positive test from any sample fell beyond 14 days of symptom onset. NGS was conducted on 18 SN and saliva samples, with phylogenetic analyses demonstrating lineages for all samples tested were Clade O (GISAID nomenclature) and lineage B.6 (PANGOLIN nomenclature). Conclusion This study supports saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further delineates the role of oropharyngeal secretions in increasing the sensitivity of testing. However, SN swabs were inferior as an alternate sample type. Our study also provides evidence that a straightforward next-generation sequencing workflow can provide direct-from-sample phylogenetic analysis for public health decision-making.

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