Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2

Hashemi, Seyed Ahmad; Khoshi, Amirhosein; Ghasemzadeh‐Moghaddam, Hamed; Ghafouri, Majid; Taghavi, Mohammadreza; Namdar-Ahmadabad, Hasan; Azimian, Amir

doi:10.1016/j.meegid.2020.104625

Cited by 11 publications

(12 citation statements)

References 17 publications

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“…Considering the importance of D614G compared with other mutations, a simple and rapid method to detect this mutation is favorable. Prior to this, PDRA has been successfully used to detect heart disease-related SNP and prostate cancer-related SNP (12)(13).…”

Section: Discussionmentioning

confidence: 99%

“…Recombinase-aided amplification (RAA) is a new isothermal amplification assay for various pathogens (11) and uses specific enzymes and proteins to quickly detect nucleic acid in less than 30 minutes at 39 °C, and it is cheaper (4.6 USD/test). Recently, a modified RAA assay, probe-directed recombinase amplification (PDRA), was developed in our laboratory for detection of SNPs (12)(13). In this study, a PDRA method for simple and quick differentiation of D614G mutation in COVID-19 was reported.…”

Section: Introductionmentioning

confidence: 99%

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Development of a PDRA Method for Detection of the D614G Mutation in COVID-19 Virus — Worldwide, 2021

Chen

Shen

et al. 2021

China CDC Weekly

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Background: COVID-19 infection is a major public health problem worldwide, and the D614G mutation enhances the infectivity of COVID-19.Methods: A probe-directed recombinase amplification (PDRA) assay was discussed to detect the D614G mutation at 39 ℃ for 30 min. The sensitivity, specificity, and reproducibility of the PDRA were evaluated by D614 and G614 recombinant plasmids. The clinical performance of PDRA assay was validated by testing of 53 previously confirmed COVID-19 positive RNAs and 10 negative samples. Direct sequencing was carried out in parallel for comparison.Result: With good reproducibility and specificity, the PDRA assay worked well with the concentration in the range of 10 3 -10 7 copies/reaction. Compared with direct sequencing as a reference, the recombinase-aided amplification (RAA) assay obtained 100% sensitivity and 100% specificity using clinical samples.Conclusions: A rapid, convenient, sensitive, and specific method to detect D614G mutation was developed, which offers a useful tool to monitor mutations in COVID-19 virus RNA.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of a PDRA Method for Detection of the D614G Mutation in COVID-19 Virus — Worldwide, 2021

Chen

Shen

et al. 2021

China CDC Weekly

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“…Another study illustrated that the Biosensing approach was also reliable in detecting the D614G mutation [ 15 ]. The PCR-RFLP method has also been described to detect the D614G mutation, but the data were ultimately misinterpreted 16 , 17 . Another possible and reliable method for detecting SNP is real-time PCR and high-resolution melting curve analysis (HRM) [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis

Gazali¹,

Nuhamunada²,

Nabilla³

et al. 2021

Heliyon

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Objectives Monitoring 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. Methods Six 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. Results The results showed that the melting temperature (T m ) of the D614 variant was 79.39 ± 0.03°C, which was slightly lower than the T m 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. Conclusions This 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.

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“…Therefore, developing inexpensive and rapid methods for identifying SNVs, such as D614G, are essential for tracking this variant by epidemiologists, molecular virologists or immunologists collaborating with clinicians to compare Covid-19 patients with SARS CoV-2 D614 and G614 variants. A previous study mistakenly developed an RFLP method for identifying another mutation at residue 615 of the spike protein ( Hashemi et al, 2020 ), but not D614G as commented by Niranji and Al-Jaf ( Niranji and Al-Jaf, 2021 ). A study highlighted the necessity of SARS CoV-2 D614G mutant using biosensing and restriction enzyme methods including Bts CI endonuclease which can cleave the wild type D614 but do not cut G614 mutant (Zhang et al, 2021) .…”

Section: Introductionmentioning

confidence: 99%

Rapid, inexpensive methods for exploring SARS CoV-2 D614G mutation

2021

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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.

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Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2

Cited by 11 publications

References 17 publications

Development of a PDRA Method for Detection of the D614G Mutation in COVID-19 Virus — Worldwide, 2021

Development of a PDRA Method for Detection of the D614G Mutation in COVID-19 Virus — Worldwide, 2021

Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis

Rapid, inexpensive methods for exploring SARS CoV-2 D614G mutation

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