Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.

Magnetic particles for separation and purification of nucleic acids

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.

https://cmr.asm.org/content/cmr/34/3/e00228-20.full.pdf

Tools and Techniques for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/COVID-19 Detection.

COVID-19 is an acute respiratory disease caused by SARS-CoV-2, which has high transmissibility. People infected with SARS-CoV-2 can develop symptoms including cough, fever, pneumonia and other complications, which in severe cases could lead to death. In addition, a proportion of people infected with SARS-CoV-2 may be asymptomatic. At present, the primary diagnostic method for COVID-19 is reverse transcription-polymerase chain reaction (RT-PCR), which tests patient samples including nasopharyngeal swabs, sputum and other lower respiratory tract secretions. Other detection methods, e.g., isothermal nucleic acid amplification, CRISPR, immunochromatography, enzyme-linked immunosorbent assay (ELISA) and electrochemical sensors are also in use. As the current testing methods are mostly performed at central hospitals and third-party testing centres, the testing systems used mostly employ large, high-throughput, automated equipment. Given the current situation of the epidemic, point-of-care testing (POCT) is advantageous in terms of its ease of use, greater approachability on the user's end, more timely detection, and comparable accuracy and sensitivity, which could reduce the testing load on central hospitals. POCT is thus conducive to daily epidemic control and achieving early detection and treatment. This paper summarises the latest research advances in POCT-based SARS-CoV-2 detection methods, compares three categories of commercially available products, i.e., nucleic acid tests, immunoassays and novel sensors, and proposes the expectations for the development of POCT-based SARS-CoV-2 detection including greater accessibility, higher sensitivity and lower costs.

Point-of-care testing detection methods for COVID-19.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global health emergency, and its gene mutation and evolution further posed uncertainty of epidemic risk. Herein, we reported a light-up CRISPR-Cas13 transcription amplification method, which enables the detection of SARS-CoV-2 and its mutated variants. Sequence specificity was ensured by both the ligation process and Cas13a/crRNA recognition, allowing us to identify viral RNA mutation. Light-up RNA aptamer allows sensitive output of amplification signals via target-activated ribonuclease activity of CRISPR-Cas13a. The RNA virus assay has been designed to detect coronavirus, SARS-CoV-2, Middle East respiratory syndrome (MERS), and SARS, as well as the influenza viruses such as, H1N1, H7N9, and H9N2. It was accommodated to sense as low as 82 copies of SARS-CoV-2. Particularly, it allowed us to strictly discriminate key mutation of the SARS-CoV-2 variant, D614G, which may induce higher epidemic and pathogenetic risk. The proposed RNA virus assays are promising for point-of-care monitoring of SARS-CoV-2 and its risking variants.

Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification.

The potential application of electrochemical biosensors in the COVID-19 pandemic: A perspective on the rapid diagnostics of SARS-CoV-2.

The pandemic of coronavirus disease 2019 (COVID-19) highlights the importance of rapid and sensitive diagnostics of viral infection that enables the efficient tracing of cases and the implementation of public health measures for disease containment. The immediate actions from both academia and industry have led to the development of many COVID-19 diagnostic systems that have secured fast-track regulatory approvals and have been serving our healthcare frontlines since the early stage of the pandemic. On diagnostic technologies, many of these clinically validated systems have significantly benefited from the recent advances in micro- and nanotechnologies in terms of platform design, analytical method, and system integration and miniaturization. The continued development of new diagnostic platforms integrating micro- and nanocomponents will address some of the shortcomings we have witnessed in the existing COVID-19 diagnostic systems. This Perspective reviews the previous and ongoing research efforts on developing integrated micro- and nanosystems for nucleic acid-based virus detection, and highlights promising technologies that could provide better solutions for the diagnosis of COVID-19 and other viral infectious diseases. With the summary and outlook of this rapidly evolving research field, we hope to inspire more research and development activities to better prepare our society for future public health crises.

Ilina Kolker Baravik

Papers

Fighting COVID-19: Integrated Micro- and Nanosystems for Viral Infection Diagnostics.