Comments on 'Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2'.
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ABSTRACT:
SUBMITTER: Niranji SS
PROVIDER: S-EPMC7709600 | biostudies-literature | 2021 Jan
REPOSITORIES: biostudies-literature
Publications
Comments on 'Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2'.
Niranji Sherko S SS Al-Jaf Sirwan M A SMA
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 20201202
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Project description:In late 2019, an outbreak of respiratory disease named COVID-19 started in the world. To date, thousands of cases of infection are reported worldwide. Most researchers focused on epidemiology and clinical features of COVID-19, and a small part of studies was performed to evaluate the genetic characteristics of this virus. Regarding the high price and low availability of sequencing techniques in developing countries, here we describe a rapid and inexpensive method for the detection of D614G mutation in SARS-CoV-2. Using bioinformatics databases and software, we designed the PCR-RFLP method for D614G mutation detection. We evaluated 144 SARS-CoV-2 positive samples isolated in six months in Northeastern Iran. Our results showed that the prevalent type is S-D in our isolates, and a small number of isolated belongs to the S-G type. Of 144 samples, 127 (88.2%) samples have belonged to type S-D, and 13 (9%) samples typed S-G. The first S-G type was detected on 2020 June 10. We have little information about the prevalence of D614G mutation, and it seems that the reason is the lack of cheap and fast methods. We hope that this method will provide more information on the prevalence and epidemiology of D614G mutations worldwide.
| S-EPMC7648195 | biostudies-literature
Project description: Not available
| S-EPMC9192182 | biostudies-literature
Project description:The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.
| S-EPMC7758719 | biostudies-literature
Project description:ObjectivesMonitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome.MethodsSix SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods.ResultsThe results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing.ConclusionsThis study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.
| S-EPMC8420086 | biostudies-literature
Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
| S-EPMC8158177 | biostudies-literature
Project description:Fast evolving of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has caused the spreading of COVID-19 disease rapidly around the globe. The mutation, especially in the gene encoding spike protein has helped the virus adapt and evade human immune system, as well as affect the efficacy of the immunizations and treatments. SARS-CoV-2 variant carrying D614G amino acid change at the spike protein is the most dominant strain in the pandemic. Therefore, efficient detection of the SARS-CoV-2 variants including D614G mutation is critical to control the COVID-19 pandemic. Herein, we report a dual synthetic mismatches CRISPR/Cas12a (dsmCRISPR) method to detect the SARS-CoV-2 D614G mutation with high sensitivity and specificity. By targeting SARS-CoV-2 D614G mutation, synthetic mismatch crRNAs were designed from -3 to +3 position around the mutation site. To improve the sensitivity and specificity, a synthetic mismatch primer with a 3'-terminal base complementary to the D614G point mutation and a mismatch next to 3'-terminal base was used to specifically amplify the D614G mutation site with higher annealing temperature. Using synthetic mismatch crRNA-(-1), a higher ratio (13.45) of the fluorescence between G614 and D614 was observed. When combined with mismatch primer to amplify D614G mutation, the fluorescence ratio of G614/D164 template detected was increased by 73.53% to 23.12. This method can detect the SARS-CoV-2 D614G mutation nucleic acid with high sensitivity, which was validated with synthetic SARS-CoV-2 D614G RNA. Therefore, the dsmCRIPSR method has significant potential to serve as a sensitive and specific assay for SARS-CoV-2 D614G detection and could be further extended for the detection of other SARS-CoV-2 variants of interest.
| S-EPMC8339451 | biostudies-literature
Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein acquired a D614G mutation early in the pandemic that confers greater infectivity and is now the globally dominant form. To determine whether D614G might also mediate neutralization escape that could compromise vaccine efficacy, sera from spike-immunized mice, nonhuman primates, and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 spike. In all cases, the G614 pseudovirus was moderately more susceptible to neutralization. The G614 pseudovirus also was more susceptible to neutralization by receptor-binding domain (RBD) monoclonal antibodies and convalescent sera from people infected with either form of the virus. Negative stain electron microscopy revealed a higher percentage of the 1-RBD "up" conformation in the G614 spike, suggesting increased epitope exposure as a mechanism of enhanced vulnerability to neutralization. Based on these findings, the D614G mutation is not expected to be an obstacle for current vaccine development.
| S-EPMC7707640 | biostudies-literature
Project description:A common mutation has occurred in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), known as D614G (A23403G). There are discrepancies in the impact of this mutation on the virus's infectivity. Also, the whole genome sequencings are expensive and time-consuming. This study aims to develop three fast economical assays for prompt identifications of the D614G mutation including Taqman probe-based real-time reverse transcriptase polymerase chain reaction (rRT PCR), an amplification refractory mutation system (ARMS) RT and restriction fragment length polymorphism (RFLP), in nasopharyngeal swab samples. Both rRT and ARMS data showed G614 mutants indicated by the presence of HEX probe and 176 bp, respectively. Additionally, the results of the RFLP data and DNA sequencings confirmed the prevalence of the G614 mutants. These methods will be important, in epidemiological, reinfections and zoonotic aspects, through detecting the G614 mutant in retro-perspective samples to track its origins and future re-emergence of D614 wild type.
| S-EPMC8286243 | biostudies-literature
Project description: Not available
| EMPIAR-10891 | biostudies-other
Project description:A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2. D614G mutation induces structural changes in the spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on S-protein structure. The introduction of the mutation orders the 630-loop structure and thereby induces global structural changes toward the cryoelectron microscopy structure of the D614G S-protein. The ordered 630-loop weakens local interactions between the 614th residue and others in contrast to disordered structures in the wild-type protein. The mutation allosterically alters global interactions between receptor-binding domains, forming an asymmetric and mobile down conformation and facilitating transitions toward up conformation. The loss of salt bridge between D614 and K854 upon the mutation generally stabilizes S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding the molecular basis of D614G mutation is crucial as it dominates in all variants of concern, including Delta and Omicron.
| S-EPMC9671695 | biostudies-literature