关键词: COVID-19 SARS-CoV-2 bioluminescence bioluminescence resonance energy transfer biosensor molecular beacon

来  源:   DOI:10.3389/fbioe.2024.1353479   PDF(Pubmed)

Abstract:
The need for the early detection of emerging pathogenic viruses and their newer variants has driven the urgent demand for developing point-of-care diagnostic tools. Although nucleic acid-based methods such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and loop-mediated isothermal amplification (LAMP) have been developed, a more facile and robust platform is still required. To address this need, as a proof-of-principle study, we engineered a prototype-the versatile, sensitive, rapid, and cost-effective bioluminescence resonance energy transfer (BRET)-based biosensor for oligonucleotide detection (BioOD). Specifically, we designed BioODs against the SARS-CoV-2 parental (Wuhan strain) and B.1.617.2 Delta variant through the conjugation of specific, fluorescently modified molecular beacons (sensor module) through a complementary oligonucleotide handle DNA functionalized with the NanoLuc (NLuc) luciferase protein such that the dissolution of the molecular beacon loop upon the binding of the viral oligonucleotide will result in a decrease in BRET efficiency and, thus, a change in the bioluminescence spectra. Following the assembly of the BioODs, we determined their kinetics response, affinity for variant-specific oligonucleotides, and specificity, and found them to be rapid and highly specific. Furthermore, the decrease in BRET efficiency of the BioODs in the presence of viral oligonucleotides can be detected as a change in color in cell phone camera images. We envisage that the BioODs developed here will find application in detecting viral infections with variant specificity in a point-of-care-testing format, thus aiding in large-scale viral infection surveillance.
摘要:
对早期检测新出现的致病性病毒及其较新变体的需求推动了对开发即时诊断工具的迫切需求。尽管已经开发了基于核酸的方法,例如逆转录定量聚合酶链反应(RT-qPCR)和环介导等温扩增(LAMP),仍然需要一个更简单和强大的平台。为了满足这一需求,作为一项原理证明研究,我们设计了一个原型——多才多艺的,敏感,快速,和具有成本效益的基于生物发光共振能量转移(BRET)的用于寡核苷酸检测(BioOD)的生物传感器。具体来说,我们设计了针对SARS-CoV-2亲本(武汉株)和B.1.617.2Delta变体的BioOD,荧光修饰的分子信标(传感器模块)通过互补寡核苷酸处理DNA官能化的NanoLuc(NLuc)荧光素酶蛋白,使得分子信标环的溶解在病毒寡核苷酸的结合将导致BRET效率降低,因此,生物发光光谱的变化.在BioOD的组装之后,我们确定了它们的动力学反应,对变体特异性寡核苷酸的亲和力,和特异性,发现它们是快速和高度具体的。此外,在病毒寡核苷酸的存在下,BioOD的BRET效率的降低可以检测为手机相机图像中颜色的变化。我们设想,这里开发的BioOD将在检测病毒感染中找到应用,并在现场护理测试格式中具有变异特异性。从而有助于大规模的病毒感染监测。
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