A Rapid and Consistent Method To Identify SARS-CoV-2 Variants By RT-PCR

Abstract Background Since 2020, the COVID-19 pandemic spread worldwide causing health, economic, and social distresses. Containment strategy relay on rapid and consistent methodology for molecular detection and characterization. The emerging variants of concern (VOCs) are currently associated with increased infectivity, and immune escape (natural defense mechanisms as well as a vaccine). Several VOCs has been detected and include lineage B.1.1.7 first identified in the UK, linage B.1.351 in South Africa, and lineage P.1 (B.1.1.28.1) in Brazil. Here we validated a rapid and low-cost technique to distinguish B.1.1.7, B.1.351 and P.1 SARS-CoV-2 variants by detecting Spike gene mutations using RT-PCR methodology. Results We recruited 77 positive patients affected by Coronavirus Disease-19 (COVID-19). Specific Real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed targeting single nucleotide polymorphisms (SNPs) to screen Spike protein mutations. All data were validated by next generation sequencing (NGS) methodology and using sequence from a public database.Among 77 COVID-19 positive samples we could discriminate with 100% of concordance all the investigated SARS-CoV-2 variants when comparing with NGS method. Conclusions PCR-based assays for identification of circulating VOCs of SARS-CoV-2 resulted in a rapid method to identify the specific SARS-CoV-2 variants allowing a better survey of the spread of the virus and its transmissibility in the pandemic phase.

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A Rapid and Consistent Method to Identify SARS-CoV-2 Variants by RT-PCR

10.21203/rs.3.rs-521517/v1 ◽

2021 ◽

Author(s):

Marco Fabiani ◽

Katia Margiotti ◽

Manuela Sabatino ◽

Antonella Viola ◽

Alvaro Mesoraca ◽

...

Keyword(s):

Defense Mechanisms ◽

Immune Escape ◽

Low Cost ◽

Gene Mutations ◽

Nucleotide Polymorphisms ◽

Rt Pcr ◽

Spike Gene ◽

Natural Defense ◽

The Uk ◽

Next Generation Sequencing Ngs

Abstract Background: Since 2020, the COVID-19 pandemic spread worldwide causing health, economic, and social distresses. Containment strategy relay on rapid and consistent methodology for molecular detection and characterization. The emerging variants of concern (VOCs) are currently associated with increased infectivity, and immune escape (natural defense mechanisms as well as a vaccine). Several VOCs has been detected and include lineage B.1.1.7 first identified in the UK, linage B.1.351 in South Africa, and lineage P.1 (B.1.1.28.1) in Brazil. Here we validated a rapid and low-cost technique to distinguish B.1.1.7, B.1.351 and P.1 SARS-CoV-2 variants by detecting Spike gene mutations using RT-PCR methodology. Results: We recruited 77 positive patients affected by Coronavirus Disease-19 (COVID-19). Specific Real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed targeting single nucleotide polymorphisms (SNPs) to screen Spike protein mutations. All data were validated by next generation sequencing (NGS) methodology and using sequence from a public database.Among 77 COVID-19 positive samples we could discriminate with 100% of concordance all the investigated SARS-CoV-2 variants when comparing with NGS method.Conclusions: PCR-based assays for identification of circulating VOCs of SARS-CoV-2 resulted in a rapid method to identify the specific SARS-CoV-2 variants allowing a better survey of the spread of the virus and its transmissibility in the pandemic phase.

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A Rapid and Low-Cost protocol for the detection of B.1.1.7 lineage of SARS-CoV-2 by using SYBR Green-Based RT-qPCR

10.1101/2021.01.27.21250048 ◽

2021 ◽

Author(s):

Fadi Abdel Sater ◽

Mahmoud Younes ◽

Hassan Nassar ◽

Paul Nguewa ◽

Kassem Hamze

Keyword(s):

Low Cost ◽

False Negative ◽

Sybr Green ◽

Reproductive Number ◽

Rt Pcr ◽

Negative Results ◽

False Negative Results ◽

New Variant ◽

Primer Sets ◽

The Uk

AbstractBackgroundThe new SARS-CoV-2 variant VUI (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit).MethodsTo control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to SARS-CoV-2 variant VUI (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile.ResultsOur results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene.ConclusionsThis technique may allow to identify patients carrying the VUI (202012/01) variant or a closely related variant, in case of shortage in sequencing.

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B.1.617.3 SARS CoV-2 spike E156G/Δ157-158 mutations contribute to reduced neutralization sensitivity and increased infectivity

10.1101/2021.10.04.463028 ◽

2021 ◽

Author(s):

Tarun Mishra ◽

Garima Joshi ◽

Atul Kumar ◽

Rishikesh Dalavi ◽

Pankaj Pandey ◽

...

Keyword(s):

Immune Evasion ◽

Neutralizing Antibodies ◽

Immune Escape ◽

Coding Region ◽

Rt Pcr ◽

Breakthrough Infection ◽

Neutralization Sensitivity ◽

Spike Gene ◽

Spike Coding ◽

The Usa

SARS CoV-2 variants raise significant concerns due to their ability to cause vaccine breakthrough infections. Here, we sequence-characterized the spike gene, isolated from a breakthrough infection, that corresponded to B.1.617.3 lineage. Delineating the functional impact of spike mutations using reporter pseudoviruses (PV) revealed that N-terminal domain (NTD)-specific E156G/Δ157-158 contributed to increased infectivity and reduced sensitivity to ChAdOx1 nCoV-19 vaccine (CovishieldTM)-elicited neutralizing antibodies. A six-nucleotide deletion (467-472) in the spike coding region introduced this change in the NTD. We confirmed the presence of E156G/Δ157-158 in the RT-PCR-positive cases concurrently screened, in addition to other circulating spike (S1) mutations like T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/Δ157-158 was present in more than 85% of the sequences reported from the USA, UK, and India in August 2021. The spike PV bearing combination of E156G/Δ157-158 and L452R further promoted infectivity and conferred immune evasion. Additionally, increased cell-to-cell fusion was observed when spike harbored E156G/Δ157-158, L452R, and E484Q, suggesting a combinatorial effect of these mutations. Notwithstanding, the plasma from a recovered individual robustly inhibited mutant spike PV, indicating the increased breadth of neutralization post-recovery. Our data highlights the importance of spike NTD-specific changes in determining infectivity and immune escape of variants.

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Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mutational Pattern in the Fourth Pandemic Phase in Greece

Current Issues in Molecular Biology ◽

10.3390/cimb44010024 ◽

2022 ◽

Vol 44 (1) ◽

pp. 329-335

Author(s):

Panagiotis Halvatsiotis ◽

Sofia Vassiliu ◽

Panagiotis Koulouvaris ◽

Kalliopi Chatzantonaki ◽

Konstantinos Asonitis ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Rural Areas ◽

Gene Mutations ◽

Threshold Value ◽

Rapid Review ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Spike Gene ◽

Copy Numbers

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%).

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Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific PCR

10.1101/2021.10.08.21264472 ◽

2021 ◽

Author(s):

Jeremy A Garson ◽

Samuel Badru ◽

Eleanor L Parker ◽

Richard S Tedder ◽

Myra O McClure

Keyword(s):

Influenza A ◽

Gene Mutations ◽

Clinical Samples ◽

Rt Pcr ◽

Screening Assay ◽

Spike Gene ◽

Specific Pcr ◽

The World ◽

Allele Specific ◽

Syncytial Virus

The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in Europe, the USA, and many other parts of the world. It is essential to monitor efficiently its spread to help guide public health policies. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. We describe here a rapid and relatively inexpensive alternative to sequencing for specific identification of the Delta variant, by application of double-mismatch allele-specific RT-PCR (DMAS-RT-PCR). The technique exploits forward and reverse allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer is enhanced by the presence of an additional mismatch located at the fourth nucleotide from the 3' end. Amplicons are detected in real-time by means of a conventional fluorescently-labelled hydrolysis probe. Specificity was assessed by testing a range of well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants of concern (Alpha, B.1.1.7 and Beta, B.1.351), and 'wild-type' SARS-CoV-2. Other respiratory viruses, including influenza A and respiratory syncytial virus, were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay, which targets the E gene. Several samples that could not be sequenced due to insufficient virus could successfully be genotyped by DMAS-RT-PCR. The method we describe would be simple to establish in any laboratory that has the ability to conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant throughout the world, and its proportional representation in any SARS-CoV-2-infected population.

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Utilization of a SARS-CoV-2 Variant Assay for the Rapid Differentiation of Omicron and Delta

10.1101/2021.12.22.21268195 ◽

2021 ◽

Author(s):

Nicholas Jacob Barasch ◽

James Iqbal ◽

Marvin Coombs ◽

Sofia Kazi ◽

Jessica Wang-Rodriguez ◽

...

Keyword(s):

Next Generation Sequencing ◽

Severe Acute Respiratory Syndrome ◽

Therapeutic Intervention ◽

Next Generation ◽

Rt Pcr ◽

Pcr Assay ◽

Spike Gene ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529), creates a diagnostic vacuum, since differentiation of Omicron from Delta relies on relatively slow next generation sequencing (NGS) technology delaying epidemiologic understanding and therapeutic intervention. The RUO SARS-CoV-2 Variant Set 1 Test (RSCov2V1) RT-PCR for detection of spike gene N501Y, E484K and del69-70 was designed to differentiate Alpha from Beta and Gamma variants. While Delta lacks these three variants, Omicron has the N501Y and del69-70 mutation. We submitted 88 samples for RSCov2V1 identifying 9 samples with the N501Y and del69-70 mutations while all other samples (79) were negative for all three variants. 9/9 samples with the del69-70 and N501Y were identified by NGS to be Omicron while 47/47 other samples assessed by NGS were confirmed to be Delta family variants. We demonstrate here that an immediately available RT-PCR assay for detection of spike gene N501Y and del69-70 can be utilized to rapidly differentiate Omicron from Delta variants in the proper epidemiologic context

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Statin-induced adverse effects – facts and genes

Orvosi Hetilap ◽

10.1556/oh.2013.29530 ◽

2013 ◽

Vol 154 (3) ◽

pp. 83-92

Author(s):

Mariann Harangi ◽

Noémi Zsíros ◽

Lilla Juhász ◽

György Paragh

Keyword(s):

Risk Factors ◽

Single Nucleotide Polymorphisms ◽

Adverse Effects ◽

Adverse Events ◽

Statin Therapy ◽

Significant Proportion ◽

Gene Mutations ◽

Nucleotide Polymorphisms ◽

Serious Adverse Events ◽

Single Nucleotide

Statin therapy is considered to be safe and rarely associated with serious adverse events. However, a significant proportion of patients on statin therapy show some degree of intolerance which can lead to decreased adherence to statin therapy. The authors summarize the symptoms, signs and frequencies of the most common statin-induced adverse effects and their most important risk factors including some single nucleotide polymorphisms and gene mutations. Also, they review the available approaches to detect and manage the statin-intolerant patients. Orv. Hetil., 2013, 154, 83–92.

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Monitoring the marine environment for small round structured viruses (SRSVS): a new approach to combating the transmission of these viruses by molluscan shellfish

Water Science & Technology ◽

10.2166/wst.1998.0498 ◽

1998 ◽

Vol 38 (12) ◽

pp. 51-56 ◽

Cited By ~ 12

Author(s):

K. Henshilwood ◽

J. Green ◽

D. N. Lees

Keyword(s):

Enteric Virus ◽

Winter Period ◽

Rt Pcr ◽

Cloning And Sequencing ◽

New Approach ◽

Human Enteric Virus ◽

Different Strains ◽

The Uk ◽

Public Health Protection

This study investigates human enteric virus contamination of a shellfish harvesting area. Samples were analysed over a 14-month period for Small Round Structured Viruses (SRSVs) using a previously developed nested RT-PCR. A clear seasonal difference was observed with the largest numbers of positive samples obtained during the winter period (October to March). This data concurs with the known winter association of gastroenteric illness due to oyster consumption in the UK and also with the majority of the outbreaks associated with shellfish harvested from this area during the study period. RT-PCR positive amplicons were further characterised by cloning and sequencing. Sequence analysis of the positive samples identified eleven SRSV strains, of both Genogroup I and Genogroup II, occurring throughout the study period. Many shellfish samples contained a mixture of strains with a few samples containing up to three different strains with both Genogroups represented. The observed common occurrence of strain mixtures may have implications for the role of shellfish as a vector for dissemination of SRSV strains. These results show that nested RT-PCR can identify SRSV contamination in shellfish harvesting areas. Virus monitoring of shellfish harvesting areas by specialist laboratories using RT-PCR is a possible approach to combating the transmission of SRSVs by molluscan shellfish and could potentially offer significantly enhanced levels of public health protection.

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Detection of SARS-CoV-2 from patient fecal samples by whole genome sequencing

Gut Pathogens ◽

10.1186/s13099-021-00398-5 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Andreas Papoutsis ◽

Thomas Borody ◽

Siba Dolai ◽

Jordan Daniels ◽

Skylar Steinberg ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Rt Pcr ◽

Whole Genome Analysis ◽

Fecal Samples ◽

Oral Transmission ◽

Study Participants ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1).

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Structural Mapping of Mutations in Spike, RdRp and Orf3a Genes of SARS-CoV-2 in Influenza Like Illness (ILI) Patients

Viruses ◽

10.3390/v13010136 ◽

2021 ◽

Vol 13 (1) ◽

pp. 136

Author(s):

Bandar Alosaimi ◽

Asif Naeem ◽

Majed F. Alghoribi ◽

Lilian Okdah ◽

Maaweya E. Hamed ◽

...

Keyword(s):

Surveillance Systems ◽

Case Definitions ◽

Structural Mapping ◽

Policy Makers ◽

Influenza Like Illness ◽

Spike Gene ◽

Ambulatory Patients ◽

Community Transmission ◽

Next Generation Sequencing Ngs ◽

Epidemiological Link

In December 2019, the emergence of SARS-CoV-2 virus in China led to a pandemic. Since both Influenza Like Illness (ILI) and COVID-19 case definitions overlap, we re-investigated the ILI cases using PCR for the presence of SARS-CoV-2 in 739 nasopharyngeal swabs collected from November 2019 to March 2020. SARS-CoV-2 RNA was found in 37 samples (5%) collected mostly during February 2020. It was followed by confirmation of evolutionary and spatial relationships using next generation sequencing (NGS). We observed that the overall incidence of ILI cases during 2019–2020 influenza season was considerably higher than previous years and was gradually replaced with SARS-CoV-2, which indicated a silent transmission among ambulatory patients. Sequencing of representative isolates confirmed independent introductions and silent transmission earlier than previously thought. Evolutionary and spatial analyses revealed clustering in the GH clade, characterized by three amino acid substitutions in spike gene (D614G), RdRp (P323L) and NS3 (Q57H). P323L causes conformational change near nsp8 binding site that might affect virus replication and transcription. In conclusion, assessment of the community transmission among patients with mild COVID-19 illness, particularly those without epidemiological link for acquiring the virus, is of utmost importance to guide policy makers to optimize public health interventions. The detection of SARS-CoV-2 in ILI cases shows the importance of ILI surveillance systems and warrants its further strengthening to mitigate the ongoing transmission of SARS-CoV-2. The effect of NS3 substitutions on oligomerization or membrane channel function (intra- and extracellular) needs functional validation.

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