Since the onset of the COVID-19 pandemic, over 610 million cases have been diagnosed and it has caused over 6.5 million deaths worldwide. The crisis has forced the scientific community to develop tools for disease control and management at a pace never seen before. The control of the pandemic heavily relies in the use of fast and accurate diagnostics, that allow testing at a large scale. The gold standard diagnosis of viral infections is the RT-qPCR. Although it provides consistent and reliable results, it is hampered by its limited throughput and technical requirements. Here, we discuss the main approaches to rapid and point-of-care diagnostics based on RT-qPCR and isothermal amplification diagnostics. We describe the main COVID-19 molecular diagnostic tests approved for self-testing at home or for point-of-care testing and compare the available options. We define the influence of specimen selection and processing, the clinical validation, result readout improvement strategies, the combination with CRISPR-based detection and the diagnostic challenge posed by SARS-CoV-2 variants for different isothermal amplification techniques, with a particular focus on LAMP and recombinase polymerase amplification (RPA). Finally, we try to shed light on the effect the improvement in molecular diagnostics during the COVID-19 pandemic could have in the future of other infectious diseases.

/pdf/the-future-of-point-of-care-nucleic-acid-amplification-i6z5vfgd.pdf

The Future of Point-of-Care Nucleic Acid Amplification Diagnostics after COVID-19: Time to Walk the Walk

The COVID-19 pandemic (caused by the SARS_CoV_2 virus) has emphasized the need for quick, easy-to-operate, reliable, and affordable diagnostic tests and devices at the Point-of-Care (POC) for homes/fields/clinics. Such tests and devices will contribute significantly to the fight against the COVID-19 pandemic and any future infectious disease epidemic. Often, academic research studies and those from industry lack knowledge of each other's developments. Here, we introduced DNA Polymerase Chain Reaction (PCR) and isothermal amplification reactions and reviewed the current commercially available POC nucleic acid diagnostic devices. In addition, we reviewed the history and the recent advancements in an effort to develop reliable, quick, portable, cost-effective, and automatic point-of-care nucleic acid diagnostic devices, from sample to result. The purpose of this paper is to bridge the gap between academia and industry and to share important knowledge on this subject.

Point-of-care nucleic acid tests: assays and devices.

Arboviruses are a diverse family of vector-borne pathogens that include members of the Flaviviridae, Togaviridae, Phenuviridae, Peribunyaviridae, Reoviridae, Asfarviridae, Rhabdoviridae, Orthomyxoviridae and Poxviridae families. It is thought that new world arboviruses such as yellow fever virus emerged in the 16th century due to the slave trade from Africa to America. Severe disease-causing viruses in humans include Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Crimean–Congo hemorrhagic fever virus (CCHFV), severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV). Numerous methods have been developed to detect the presence of these pathogens in clinical samples, including enzyme-linked immunosorbent assays (ELISAs), lateral flow assays (LFAs) and reverse transcriptase–polymerase chain reaction (RT-PCR). Most of these assays are performed in centralized laboratories due to the need for specialized equipment, such as PCR thermal cyclers and dedicated infrastructure. More recently, molecular methods have been developed which can be performed at a constant temperature, termed isothermal amplification, negating the need for expensive thermal cycling equipment. In most cases, isothermal amplification can now be carried out in as little as 5–20 min. These methods can potentially be used as inexpensive point of care (POC) tests and in-field deployable applications, thus decentralizing the molecular diagnosis of arboviral disease. This review focuses on the latest developments in isothermal amplification technology and detection techniques that have been applied to arboviral diagnostics and highlights future applications of these new technologies.

https://doi.org/10.3390/microorganisms11051159

Latest Advances in Arbovirus Diagnostics

Food contaminants have consistently posed a serious threat to human health and have caused various food safety problems. The most common techniques currently used for the detection of food contaminants, include instrumental and immunoassay methods; however, these traditional methods have shown some limitations when applied to the analysis of food contaminants. Therefore, in recent years, more and more researchers have been devoted to the development of rapid, reliable, sensitive, and suitable detection techniques for the detection of food contaminants in food matrices. In this review, we first classify the analytical methods proposed in recent years into colorimetric methods, fluorescence methods, photoelectrochemical methods, electrochemical methods, nucleic acid amplification methods, and other methods, and illustrate their detection principles. In addition, the paper highlights the strategies and recent research advances for each method through typical application examples. Finally, the future perspectives of these analytical methods are presented. We hope that this review will provide researchers with valuable insights to guide the sensitive detection of food contaminants.

Detection of food contaminants: A review of established rapid analytical techniques and their applications and limitations

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

Current Trends in RNA Virus Detection via Nucleic Acid Isothermal Amplification-Based Platforms

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods—reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)—combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10–20 min, whereas for RT-LAMP, the LOD was 30–300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.

/pdf/detection-of-sars-cov-2-using-reverse-transcription-helicase-29xk5r28.pdf

Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay

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Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay