A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain.
A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain.
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DOI：
10.3390/life11101015
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发表时间：
2021-09-27
期刊：
影响因子：
--
通讯作者：
Dovas CI
中科院分区：
文献类型：
--
作者：
Chaintoutis SC;Chassalevris T;Balaska S;Mouchtaropoulou E;Tsiolas G;Vlatakis I;Tychala A;Koutsioulis D;Argiriou A;Skoura L;Dovas CI
导致SARS-CoV-2刺突蛋白受体结合域(RBD)氨基酸替换的突变与各自的变异体Alpha、Beta、Gamma或Delta的传播性增强和免疫逃逸有关。因此，迅速查明上述令人关切的变种及其与其他变种的区别对于公共卫生干预具有重要意义。为此，建立了一种使用四个锁定核酸(LNA)修饰的TaqMan探针的一步实时RT-PCR方法，以定位与尖峰蛋白RBD受体结合基序(RBM)中第478、484和501位氨基酸替换相关的特征突变。该区域包含与ACE2结合的SARS-CoV-2的大部分接触残基。一种新的策略，使用不可扩展的LNA寡核苷酸阻断剂，可以减少探针的非特异性杂交，增加了在多重PCR中检查的不同突变位点的数量。对获得的不同荧光信号的组合分析使人们能够初步区分令人担忧的SARS-CoV-2变种。该方法灵敏，Delta变异体的LOD为263拷贝/反应，Beta变异体的LOD为170拷贝/反应，扩增效率为91%，对所有靶标的线性范围为>；5log10拷贝/反应。使用已知的SARS-CoV-2阳性和阴性的人和动物样本进行检测的验证表明，它能够正确地识别目标突变并初步确定SARS-CoV-2变种的特征。使用LNA寡核苷酸阻滞剂进行突变分型的新方法可以被修改为针对RBM中四个不同位置的签名突变，并进一步扩大检测到的变异的范围。
Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD. This region contains most contacting residues of SARS-CoV-2 that bind to ACE2. A novel strategy employing the use of non-extendable LNA oligonucleotide blockers that can reduce non-specific hybridization of probes increased the number of different mutated sites examined in a multiplex PCR. The combinatory analysis of the different fluorescence signals obtained enabled the preliminary differentiation of SARS-CoV-2 variants of concern. The assay is sensitive with a LOD of 263 copies/reaction for the Delta variant, 170 copies/reaction for the Beta variant, amplification efficiencies > 91% and a linear range of >5 log10 copies/reaction against all targets. Validation of the assay using known SARS-CoV-2-positive and negative samples from humans and animals revealed its ability to correctly identify the targeted mutations and preliminary characterize the SARS-CoV-2 variants. The novel approach for mutation typing using LNA oligonucleotide blockers can be modified to target signature mutations at four different sites in the RBM and further expand the range of variants detected.
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影响因子：
2.9
作者：
Owczarzy, Richard;You, Yong;Groth, Christopher L.;Tataurov, Andrey V.
通讯作者：
Tataurov, Andrey V.
影响因子：
6.7
作者：
Chaintoutis SC;Thomou Z;Mouchtaropoulou E;Tsiolas G;Chassalevris T;Stylianaki I;Lagou M;Michailidou S;Moutou E;Koenen JJH;Dijkshoorn JW;Paraskevis D;Poutahidis T;Siarkou VI;Sypsa V;Argiriou A;Fortomaris P;Dovas CI
通讯作者：
Dovas CI
影响因子：
3.1
作者：
Chaintoutis SC;Chassalevris T;Tsiolas G;Balaska S;Vlatakis I;Mouchtaropoulou E;Siarkou VI;Tychala A;Koutsioulis D;Skoura L;Argiriou A;Dovas CI
通讯作者：
Dovas CI
影响因子：
4.1
作者：
Lee, Seung-Tae;Kim, Ji-Youn;Ki, Chang-Seok
通讯作者：
Ki, Chang-Seok
影响因子：
4.3
作者：
Chaintoutis, Serafeim C.;Siarkou, Victoria I.;Dovas, Chrysostomos I.
通讯作者：
Dovas, Chrysostomos I.