RESUMEN
The pandemic of severe acute respiratory syndrome 2 (SARS-CoV-2) revealed the necessity of diagnosis of the infected people to prevent the prevalence infection cycle. Many commercial pathogen diagnosis methods are based on the detection of genomic materials. Isothermal amplification methods such as loop-mediated-isothermal amplification (LAMP) are the method of choice in these cases. Reverse transcription steps are efficiently coupled to LAMP for the detection of pathogens with genomic RNAs such as SARS-CoV-2. Many detection systems for LAMP include fluorescent readout systems. Although such systems result in desirable limits of detection, the need for special instrumentation is the main dispute of such systems to become real point of care assays. In contrast, colorimetric detection methods would reduce costs and improve the applicability of the system. In this study one-step reverse transcription-LAMP reaction was established that enables visual detection of the SARS-CoV-2 genome. Nasopharyngeal RNA samples were first validated by reverse transcription quantitative polymerase chain reaction and then subjected to RT-LAMP. To lower the cost associated with the readout system equipment, malachite green (MG) was used. The color change of MG to blue allowed visual detection of the virus. Firstly, experiments were set up as two-step RT-LAMP reaction to identify the best primer sets. In addition, MG concentration was optimized with the significant colorimetric signal for the positive samples. Next, a one-step colorimetric method was developed for the detection of SARS-CoV-2 based on MG color shift in 2 h.
RESUMEN
The pandemic of severe acute respiratory syndrome 2 (SARS-CoV-2) revealed the necessity of diagnosis of the infected people to prevent the prevalence infection cycle. Many commercial pathogen diagnosis methods are based on the detection of genomic materials. Isothermal amplification methods such as loop-mediated-isothermal amplification (LAMP) are the method of choice in these cases. Reverse transcription steps are efficiently coupled to LAMP for the detection of pathogens with genomic RNAs such as SARS-CoV-2. Many detection systems for LAMP include fluorescent readout systems. Although such systems result in desirable limits of detection, the need for special instrumentation is the main dispute of such systems to become real point of care assays. In contrast, colorimetric detection methods would reduce costs and improve the applicability of the system. In this study one-step reverse transcription-LAMP reaction was established that enables visual detection of the SARS-CoV-2 genome. Nasopharyngeal RNA samples were first validated by reverse transcription quantitative polymerase chain reaction and then subjected to RT-LAMP. To lower the cost associated with the readout system equipment, malachite green (MG) was used. The color change of MG to blue allowed visual detection of the virus. Firstly, experiments were set up as two-step RT-LAMP reaction to identify the best primer sets. In addition, MG concentration was optimized with the significant colorimetric signal for the positive samples. Next, a one-step colorimetric method was developed for the detection of SARS-CoV-2 based on MG color shift in 2 h.
RESUMEN
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.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Simulación del Acoplamiento Molecular , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Replicación Viral/genética , Metiltransferasas/genética , Péptidos/farmacología , Antivirales/farmacología , ARN/farmacología , Exorribonucleasas/genética , Exorribonucleasas/químicaRESUMEN
Primer exchange reaction (PER) is an emergent method for non-templated synthesis of single stranded DNA molecules. PER has been shown to be effective in cell imaging systems and for detection of macromolecules. A particular application of PER is to detect a specific target nucleic acid. To this endeavor, two coupled DNA hairpins, a detector and an amplifier, play in accordance to extend a target nucleic acid with a concatemer DNA sequence. Here we introduced unified-amplifier based primer exchange reaction (UniAmPER) that beneficially extends the target by a unified-amplifier. The unified-amplifier operates as both detector and amplifier hairpins. The extension resulted in synthesis of concatemer G-rich sequences. The G-rich sequences were expected to form G-quadruplex (GQ) structures. Presence of the GQ structures were investigated by peroxidase activity of GQs in presence of hemin, H2°2 and 3,3',5,5'-Tetramethylbenzidine (TMB) as well as by fluorescence signal generation upon intercalation of thioflavin T (ThT). The presented unified-amplifier in this study facilitates application of PER systems for development of colorimetric or fluorogenic biosensors. As a proof of principle, the method has been applied for detection of reversely transcribed cDNAs from clinical SARS-CoV-2 samples.