Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis.

Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

Pathogenic viruses: Molecular detection and characterization.

Pathogenic viruses are viruses that can infect and replicate within human cells and cause diseases. The continuous emergence and re-emergence of pathogenic viruses has become a major threat to public health. Whenever pathogenic viruses emerge, their rapid detection is critical to enable implementation of specific control measures and the limitation of virus spread. Further molecular characterization to better understand these viruses is required for the development of diagnostic tests and countermeasures. Advances in molecular biology techniques have revolutionized the procedures for detection and characterization of pathogenic viruses. The development of PCR-based techniques together with DNA sequencing technology, have provided highly sensitive and specific methods to determine virus circulation. Pathogenic viruses potentially having global catastrophic consequences may emerge in regions where capacity for their detection and characterization is limited. Development of a local capacity to rapidly identify new viruses is therefore critical. This article reviews the molecular biology of pathogenic viruses and the basic principles of molecular techniques commonly used for their detection and characterization. The principles of good laboratory practices for handling pathogenic viruses are also discussed. This review aims at providing researchers and laboratory personnel with an overview of the molecular biology of pathogenic viruses and the principles of molecular techniques and good laboratory practices commonly implemented for their detection and characterization.

Molecular biology of coronaviruses: current knowledge.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) late December 2019 in Wuhan, China, marked the third introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. The constant spillover of coronaviruses from natural hosts to humans has been linked to human activities and other factors. The seriousness of this infection and the lack of effective, licensed countermeasures clearly underscore the need of more detailed and comprehensive understanding of coronavirus molecular biology. Coronaviruses are large, enveloped viruses with a positive sense single-stranded RNA genome. Currently, coronaviruses are recognized as one of the most rapidly evolving viruses due to their high genomic nucleotide substitution rates and recombination. At the molecular level, the coronaviruses employ complex strategies to successfully accomplish genome expression, virus particle assembly and virion progeny release. As the health threats from coronaviruses are constant and long-term, understanding the molecular biology of coronaviruses and controlling their spread has significant implications for global health and economic stability. This review is intended to provide an overview of our current basic knowledge of the molecular biology of coronaviruses, which is important as basic knowledge for the development of coronavirus countermeasures.