ABSTRACT
Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.
Subject(s)
COVID-19 , Colorimetry , Freeze Drying , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral , SARS-CoV-2 , Humans , COVID-19/diagnosis , COVID-19/virology , SARS-CoV-2/isolation & purification , SARS-CoV-2/genetics , Colorimetry/methods , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods , RNA, Viral/analysis , RNA, Viral/genetics , Sensitivity and Specificity , Reagent Kits, Diagnostic/standards , COVID-19 Nucleic Acid Testing/methodsABSTRACT
Leukemia is a type of cancer that affects white blood cells. In this disease, immature blood cells undergo genetic mutations, leading to excessive replication and reduced cell death compared to healthy cells. In cancer, there may be the activation of oncogenes and the deactivation of tumor suppressor genes that control certain cellular functions. Despite the undeniable contribution to the patient's recovery, conventional cancer treatments may have some not-so-beneficial effects. In this case, gene therapy appears as an alternative to classical treatments. Gene therapy delivers genetic material to cells to replace or modify dysfunctional genes, a safe method for neoplasms. One of the types of nucleic acids explored in gene therapy is microRNA (miRNA), a group of endogenous, non-proteincoding, small single-stranded RNA molecules involved in the regulation of gene expression, cell division, differentiation, angiogenesis, migration, apoptosis, and carcinogenesis. This review aims to bring together the most recent advances found in the literature on cancer gene therapy based on microRNAs in the oncological context, focusing on leukemia.
Subject(s)
Leukemia , MicroRNAs , Neoplasms , Humans , MicroRNAs/genetics , MicroRNAs/therapeutic use , MicroRNAs/metabolism , Neoplasms/genetics , Neoplasms/therapy , Neoplasms/pathology , Leukemia/genetics , Leukemia/therapy , Genetic Therapy/methods , Models, Theoretical , Gene Expression Regulation, NeoplasticABSTRACT
Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.
