[Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the Spike gene by Sanger].
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ABSTRACT:
SUBMITTER: Murillo E
PROVIDER: S-EPMC9108067 | biostudies-literature | 2022 May
REPOSITORIES: biostudies-literature
Publications
[Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger].
Murillo Enderson E Palacio-Rua Katherine K Afanador-Ayala Carlos C García-Correa Juan Felipe JF Zuluaga Andrés F AF
Enfermedades infecciosas y microbiologia clinica 20220516 5
<h4>Introduction</h4>The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process ...[more]
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Project description:IntroductionThe emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike.MethodsFifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms.ResultsBoth methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study.ConclusionThe different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.
| S-EPMC9574460 | biostudies-literature
Project description:The global demand for rapid identification of circulating SARS-CoV-2 variants of concern has led to a shortage of commercial kits. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol to identify circulating SARS-CoV-2 (variants of concern). Sets of primers flanking the SARS-CoV-2 spike gene were designed, verified and then validated using 282 nasopharyngeal positive samples for SARS-CoV-2. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole-genome sequencing of the same samples. Out of 282 samples, 123 contained the alpha variant, 78 beta and 13 delta, which were indicted using in-house primers and next-generation sequencing; the numbers of variants found were 100% identical to the reference genome. This protocol is easily adaptable for detection of emerging variants during the pandemic.
| S-EPMC9937032 | biostudies-literature
Project description:BackgroundThe evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists, giving rise to new variants characterized by mutations in the spike protein. However, public data regarding the virus's evolutionary trend is not widely available after the downgrade of coronavirus disease 2019(COVID-19). Therefore, this study aimed to investigate the applicability of an in-house Sanger-based method for identifying SARS-CoV-2 variants, particularly focusing on newly emerged Omicron variants, and updating the epidemiology of COVID-19 during the 8th wave in Hiroshima Prefecture.ResultsA total of 639 saliva samples of individuals who had tested positive for COVID-19, received from Hiroshima City Medical Association Clinical Laboratory Center between February 01, 2023, and March 12, 2024, were included in the study. SARS-CoV-2 variants were identified in 69.3% (443/639) with the mean viral titer 2 × 106 copies/mL, and high viral titer in Omicron variant XBC.1.6* (5 × 108 copies/mL) using RT-qPCR. By partial Spike gene-based sequencing using the Sanger Sequencing strategy, Omicron sub-lineages XXB.1, BA.5, and EG.1 were identified during different periods. A comprehensive phylogenetic analysis of 7383 SARS-CoV-2 strains retrieved from GISAID, collected in Hiroshima from the onset of the COVID-19 pandemic in early 2020 until July 2024, revealed the dynamic evolution of SARS-CoV-2 variants over time. The study found a similar pattern of variant distribution between the full genomes from GISAID, and the partial genomes obtained from our screening strategy during the same period.ConclusionsOur study revealed that all SARS-CoV-2 viruses circulated in Hiroshima were Omicron variants and their sub-lineages during the 8th wave outbreak in Hiroshima. Persistent molecular surveillance of SARS-CoV-2 is needed for the decision-making and strategic planning of the public promptly. Our study added evidence for the usefulness of SARS-CoV-2 spike gene partial sequencing-based SARS-CoV-2 variant identification strategy for mass screening and molecular surveillance even though the evolution of newly emerged various SARS-CoV-2 Omicron variants.
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Project description:This study aimed to develop the feasible and effective universal screening strategy of the notable SARS-CoV-2 variants by Sanger Sequencing Strategy and then practically applied it for mass screening in Hiroshima, Japan. A total of 734 samples from COVID-19 confirmed cases in Hiroshima were screened for the notable SARS-CoV-2 variants (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, C.37, B.1.1.529, etc.). The targeted spike region is amplified by nested RT-PCR using in-house designed primer set hCoV-Spike-A and standard amplification protocol. Additionally, randomly selected 96 samples were also amplified using primer sets hCoV-Spike-B and hCoV-Spike-C. The negative amplified samples were repeated for second attempt of amplification by volume-up protocol. Thereafter, the amplified products were assigned for Sanger sequencing using corresponding primers. The positive amplification rate of primer set hCoV-Spike-A, hCoV-Spike-B and hCoV-Spike-C were 87.3%, 83.3% and 93.8% respectively for standard protocol and increased to 99.6%, 95.8% and 96.9% after second attempt by volume-up protocol. The readiness of genome sequences was 96.9%, 100% and 100% respectively. Among 48 mutant isolates, 26 were B.1.1.7 (Alpha), 7 were E484K single mutation and the rest were other types of mutation. Moreover, 5 cluster cases with single mutation at N501S were firstly reported in Hiroshima. This study indicates the reliability and effectiveness of Sanger sequencing to screen large number of samples for the notable SARS-CoV-2 variants. Compared to the Next Generation Sequencing (NGS), our method introduces the feasible, universally applicable, and practically useful tool for identification of the emerging variants with less expensive and time consuming especially in those countries where the NGS is not practically available. Our method allows not only to identify the pre-existing variants but also to examine other rare type of mutation or newly emerged variants and is crucial for prevention and control of pandemic.
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Project description:The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.
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Project description:Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID50) values of 1.4 × 102 TCID50/mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.
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Project description:Molecular surveillance of the new coronavirus through new genomic sequencing technologies revealed the circulation of important variants of SARS-CoV-2. Sanger sequencing has been useful in identifying important variants of SARS-CoV-2 without the need for whole-genome sequencing. A sequencing protocol was constructed to cover a region of 1000 base pairs, from a 1120 bp product generated after a two-step RT-PCR assay in samples positive for SARS-CoV-2. Consensus sequence construction and mutation identification were performed. Of all 103 samples sequenced, 69 contained relevant variants represented by 20 BA.1, 13 delta, 22 gamma, and 14 zeta, identified between June 2020 and February 2022. All sequences found were aligned with representative sequences of the variants. Using the Sanger sequencing methodology, we were able to develop a more accessible protocol to assist viral surveillance with a more accessible platform.
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Project description:Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.
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Project description: Not available
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