A Rapid and Consistent Method To Identify SARS-CoV-2 Variants By RT-PCR (original) (raw)
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Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which c...
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The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of ...
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The ongoing coronavirus disease 2019 (COVID-19) pandemic represents one of the most exigent threats of our lifetime to global public health and economy. As part of the pandemic, from January 10 to March 10, 2020, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) began to spread in Hefei (Anhui Province, China) with a total of 174 confirmed cases of COVID-19. During this period, we were able to gather critical information on the transmission and evolution of pathogens through genomic surveillance. Particularly, the objective of our study was to track putative variants of SARS-CoV-2 circulating in Hefei for the first time and contribute to the global effort toward elucidating the molecular epidemic profile of the virus. Patients who showed symptoms of COVID-19 were routinely tested for SARS-CoV-2 infections via RT-PCR at the First Affiliated Hospital of Anhui Medical University. Whole-genome sequencing was performed on 97 clinical samples collected from 29 confirmed COVID-1...
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With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses1–3, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a “spike gene target failure” (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike Δ69-70, such as B.1.351 (also 501Y.V2) detected in South Africa6 and P.1 (also 501Y.V3) recently detected in Brazil7. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open source PCR...
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Delta and Omicron variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are remarkably contagious, and have been recognized as variants of concern (VOC). The acquisition of spontaneous substitutions or insertion-deletion mutations (indels) in the spike protein-encoding gene substantially increases the binding a nity of the receptor binding domain (RBD)-hACE2 complex and upsurges the transmission of both variants. In this study, we analyzed thousands of genome sequences representing 30 different SARS-CoV-2 variants and identi ed the Delta and Omicron variants speci c nucleic acid signatures in the spike gene. Based on the variant-speci c nucleic acid sequences, we synthesized different oligos and optimized a multiplex PCR (mPCR) assay that can identify and differentiate the Delta and Omicron variants of SARS-CoV-2. We further extended our work on this mPCR and translated it into a dipstick assay by adding a tag linker sequence to the 5' end of the forward primer and biotin to the 3' end of the oligos. Streptavidincoated latex beads and the dipstick imprinted with a probe for the tag linker sequence in the test strips were used for the detection assay. Our dipstick-based assay, developed as a rapid point-of-care test for identifying and differentiating SARS-CoV-2 variants has the potential to be used in low-resource settings and scaled up to the population level.
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The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or wholegenome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.
Perspective Chapter: SARS-CoV-2 Variants -Two Years Post-Onset of the Pandemic
Intech Open, 2022
Since the pandemic began in China in December 2019, thousands of variants of SARS-CoV-2 have emerged globally since late 2020. The World Health Organization (WHO) defined the SARS-CoV-2 variant of concern (VOC) as a variant with increased transmissibility, virulence, and decreased response to available diagnostics, vaccines, and therapeutics. Areas of the emerging variant of concern arise from countries like the United Kingdom, South Africa, Brazil, and India. These mutations carry a lineage from N501Y, D614G, N439K, Y453F, and others, which are globally dominated by clades 20A, 20B, and 20C. SARS-CoV-2 VOC emerged after 11 months of evolution since the onset through massive human-to-human transmission with five major VOCs recognized by the WHO, namely Alpha, Beta, Gamma, Delta, and Omicron. Their emergence could be attributed to changing immunological dynamics in the human population, which has resulted in resistance or escape from neutralizing antibodies, or to mutations and/or recombinations that increase transmission or pathogenicity. This literature review intends to identify and report on SARS-CoV-2 variants that have evolved two years post-onset of the pandemic and their disease implications.
Journal of Pure and Applied Microbiology, 2021
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus (CoV) disease 2019 (COVID-19). This study compared the genome, mutations, and infectivity/ transmissibility of SARS-CoV-2 with selected betacoronaviruses (beta-CoVs). This study further examined the origin, risk factors, and outbreaks caused by beta-CoVs. We searched the following databases for relevant studies: PubMed, Google Scholar, and the World Health Organization COVID-19 database. A close relationship between SARS-CoV-2 and SARS bat-like CoV RaTG13 (98.9%) was found at the amino acid level, followed by pangolin CoVs. Non-synonymous mutations occur at high frequencies in the open reading frame (ORF) 1ab, spike (S) protein, and nucleocapsid. Mutations P323L and D614G in the RNA-dependent RNA polymerase (RdRp) and S protein, respectively, occur at a high frequency globally. Mutations at position 3037 in the nonstructural protein (Nsp) 3, 14408 (RdRp), and 23403 (S) confer transmissibility to SARS-CoV-2. SARS-CoV-2 has higher infectivity and transmissibility than SARS-CoV, which shares the same receptor. Although bats are confirmed reservoirs, intermediate hosts are currently unknown. Smoking, old age, diabetes, cardiovascular diseases, and hypertension have all been associated with COVID-19. Within six months of its outbreak, COVID-19 was reported in all countries worldwide, whereas SARS was reported in 28 countries and Middle East respiratory syndrome (MERS) in 5 countries. However, the fatality rate of MERS (65%) was higher than that of COVID-19 (4.9%) and SARS (6.6%). Identifying the SARS-CoV-2 intermediate hosts will help prevent future outbreaks. Attention should be given to the pangolin CoVs. Variations in the S gene may confer transmissibility and infectivity.
Rapid screening method for the detection of SARS-CoV-2 variants of concern
Journal of Clinical Virology, 2021
Background: Comprehensive and up-to-date monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) is crucial as these are characterized by their increased transmissibility, immune evasion and virulence. Objectives: To describe the wide-scale implementation of a reverse transcriptase polymerase chain reaction (RT-PCR) multiple variants assay with melting curve analysis as a routine procedure. Study design: We prospectively performed multiple variants RT-PCR on consecutive SARS-CoV-2 RT-PCR positive samples from patients, healthcare workers and nursing home residents from our hospital catchment area. This technique was implemented in our automated Roche FLOW system with a turnaround time of 6 h. Results: Between February 1 and May 2, 2021, 989 samples were tested by the variant RT-PCR. Our method was validated by comparison of variant RT-PCR to whole genome sequencing testing. We observed an increase over time in the proportion of UK variant that became the dominant variant, and the concurrent emergence of the South-African and Brazilian variants. Prompt public health responses for infection control were possible because of this rapid screening method, resulting in early detection and reduction of unnoticed spread of VOC as early as possible. Conclusion: A variant RT-PCR with additional melting curve analyses is a feasible, rapid and efficient screening strategy that can be implemented in routine microbiological laboratories.