Articles
Publication Date: 2025
Analytical Methods (17599679)17(45)pp. 9244-9253
In recent years, thioflavin T (ThT) has been applied as an intercalating fluorescence probe in the development of biosensors. One approach is based on monitoring the fluorescent signal of ThT upon binding to a G-quadruplex (GQ). GQ is a structure that is formed from guanine-rich sequences. The sequences may be synthesized during isothermal or cyclic amplifications. The ThT-based fluorescent signal generation has been combined with various isothermal amplification methods. However, to the best of our knowledge, it has not yet been studied in combination with cyclic amplifications, i.e., polymerase chain reaction (PCR). PCR has benefits that has led to success in many commercial and FDA-approved diagnostic kits; however, the traditional qPCR methods that apply TaqMan technology suffer from challenges such as high price and dependence on sophisticated instruments. The current commercial qPCR kits are limited in their wider application by general public health authorities upon their price and availability. Thus, developing new amplification-based nucleic acid tests (NATs) was portrayed to be necessary to facilitate an improvement in public health. Here, we present a fluorimetric analysis of PCR products upon incorporating thioflavin T into the synthesized GQ as a new readout system for detecting a viral RNA. The PCR primers were designed with a reverse complementary sequence of a GQ. The system's efficiency was validated in one-pot, one-step reactions for detecting the presence of a model virus, i.e., SARS-CoV-2 genome in samples taken from individuals with various viral loads.
Publication Date: 2025
Biosensors and Bioelectronics (0956-5663)275
Emergence of recent pandemics/endemics e.g. COVID-19 and Dengue fever, demonstrated the necessity of development of strategies for swift adaptation of present biosensor for detection of the new emerging pathogens. However, development of a biosensor for a new target is time- and labor-consuming. In this study, we aimed to integrate the primer exchange reaction (PER), an isothermal technique that extends an initiator DNA with a user-defined single-stranded DNA tail, with bipolar electrochemistry. This integration led to the development of a universal biosensor, termed ViPER. We demonstrated the utility of the developed system to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic RNA as a model. The genomic RNA was reverse transcribed to a short cDNA and was tailed with a universal tag, consequently, the tagged cDNA was applied to an electrochemiluminescence integrated bipolar electrochemical biosensor (BPE-ECL). ECL signals were recorded using a digital camera and analyzed by ImageJ. The platform demonstrated a linear response over a wide dynamic range of 10−7-10−17 M for the target nucleic acid with a detection limit of 2.31 × 10−17 M for synthetic targets. The biosensor could also successfully discriminate between biological RNA samples from infected and non-infected individuals. This study introduces the potential of DNA-based visual biosensors for detecting single-stranded RNAs in low-equipped environments, and it holds promises for further development of an ultrasensitive method for various human RNA-based viral pathogens. Moreover, we can design a platform with a predetermined DNA probe sequence for a vast variety of different targets, simply by changing the PER input. © 2025 Elsevier B.V.
Publication Date: 2023
Scientific Reports (20452322)13(1)
In recent years, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the cause of the coronavirus disease (COVID-19) global pandemic, and its variants, especially those with higher transmissibility and substantial immune evasion, have highlighted the imperative for developing novel therapeutics as sustainable solutions other than vaccination to combat coronaviruses (CoVs). Beside receptor recognition and virus entry, members of the SARS-CoV-2 replication/transcription complex are promising targets for designing antivirals. Here, the interacting residues that mediate protein–protein interactions (PPIs) of nsp10 with nsp16 and nsp14 were comprehensively analyzed, and the key residues’ interaction maps, interaction energies, structural networks, and dynamics were investigated. Nsp10 stimulates both nsp14’s exoribonuclease (ExoN) and nsp16’s 2′O-methyltransferase (2′O-MTase). Nsp14 ExoN is an RNA proofreading enzyme that supports replication fidelity. Nsp16 2′O-MTase is responsible for the completion of RNA capping to ensure efficient replication and translation and escape from the host cell’s innate immune system. The results of the PPIs analysis proposed crucial information with implications for designing SARS-CoV-2 antiviral drugs. Based on the predicted shared protein–protein interfaces of the nsp16-nsp10 and nsp14-nsp10 interactions, a set of dual-target peptide inhibitors was designed. The designed peptides were evaluated by molecular docking, peptide–protein interaction analysis, and free energy calculations, and then further optimized by in silico saturation mutagenesis. Based on the predicted evolutionary conservation of the interacted target residues among CoVs, the designed peptides have the potential to be developed as dual target pan-coronavirus inhibitors. © 2023, The Author(s).
Purpose and methods: The emergence of coronavirus disease 2019 (COVID-19) has once again affirmed the significant threat of respiratory infections to global public health and the utmost importance of prompt diagnosis in managing and mitigating any pandemic. The nucleic acid amplification test (NAAT) is the primary detection method for most pathogens. Loop‐mediated isothermal amplification (LAMP) is a rapid, simple, sensitive, and specific epitome of isothermal NAAT performed using a set of four to six primers. Primer design is a fundamental step in LAMP assays, with several complexities and experimental screening requirements. To address this challenge, an online database is presented here. Its workflow comprises three steps: literature aggregation, data curation, and database and website implementation. Results: LAMPPrimerBank (https://lampprimerbank.mathematik.uni-marburg.de) is a manually curated database dedicated to experimentally validated LAMP primers, their peculiarities of assays, and accompanying literature, with a primary emphasis on respiratory pathogens. LAMPPrimerBank, with its user-friendly web interface and an open application programming interface, enables the accelerated and facile exploration, comparison, and exportation of LAMP primer sequences and their respective information from the massively scattered literature. LAMPPrimerBank currently comprises LAMP primers for diagnosing viral, bacterial, and fungal respiratory pathogens. Additionally, to address the challenge of false-positive results generated by nonspecific amplifications, LAMPPrimerBank computationally predicted and visualized the sizes of LAMP products for recorded primer sets in the database. Conclusion: LAMPPrimerBank, as a pioneering database in the rapidly expanding field of isothermal NAAT, endeavors to confront the two challenges of the LAMP: primer design and discrimination of false-positive results. © 2023, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.
