Current trends in large‐scale viral surveillance methods in mosquitoes

Vector‐borne and zoonotic infectious diseases are serious public health concerns that affect approximately half of the world's population. In particular, arthropod‐borne viruses (arboviruses) have contributed to more mortality and morbidity worldwide with the emergence of dengue, chikungunya, yellow fever, and Zika virus diseases. The infections have scaled up due to urbanization, globalization, and international mobility. Traditionally, the spread of mosquito‐borne viral diseases to humans was considered a low health priority concern. However, their categorization as emerging infectious diseases and public health emergencies of international concern has heightened the attention given by the government, academia, research, and industry for the development of timely, cost‐efficient, and sustainable solutions. The urgency has increased in the wake of global climate change. The focus on effective interventions includes epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants. In this review, we discuss the etiology and predisposition of mosquito‐borne viruses that are detrimental to public health and economically damaging when disseminated as epidemics. We focus on the large‐scale virus surveillance methods with special reference to innovations and interventions in molecular detection science and technologies that include viral nucleic acid isolation, polymerase chain reaction (PCR)‐based diagnostics, and high‐throughput sequencing technologies. In addition, we discuss the development of a viral RNA extraction and PCR‐based diagnostic kit (Invirustech) that can extract viral RNA from mosquitoes with verified applications in PCR‐based molecular diagnostics of Pan‐flavivirus.     

Multiple strategies to improve sensitivity,speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

Background  

In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).     

Molecular diagnostics: the changing culture of medical microbiology

Diagnostic molecular biology is arguably the fastest growing area in current laboratory-based medicine. Growth of the so called 'omics' technologies has, over the last decade, led to a gradual migration away from the 'one test, one pathogen' paradigm, toward multiplex approaches to infectious disease diagnosis, which have led to significant improvements in clinical diagnostics and ultimately improved patient care.     

Quantitation of viral load using real-time amplification techniques

Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA)-based systems combined with several detection strategies are being employed in a clinical diagnostic setting. The importance of these assays in disease management is still in an exploration phase. Although these technologies have the implicit capability of accurately measuring DNA and RNA in clinical samples, issues related to standardization and quality control must be resolved to enable routine implementation of these technologies in molecular diagnostics.     

DNA microarrays in the clinic: infectious diseases

We argue that the most-promising area of clinical application of microarrays in the foreseeable future is the diagnostics and monitoring of infectious diseases. Microarrays for the detection and characterization of human pathogens have already found their way into clinical practice in some countries. After discussing the persistent, yet often underestimated, importance of infectious diseases for public health, we consider the technologies that are best suited for the detection and clinical investigation of pathogens. Clinical application of microarray technologies for the detection of mycobacteria, Bacillus anthracis, HIV, hepatitis and influenza viruses, and other major pathogens, as well as the analysis of their drug-resistance patterns, illustrate our main thesis.     

Anti-glycan monoclonal antibodies: Basic research and clinical applications

Anti-glycan monoclonal antibodies have important applications in human health and basic research. Therapeutic antibodies that recognize cancer- or pathogen-associated glycans have been investigated in numerous clinical trials, resulting in two FDA-approved biopharmaceuticals. Anti-glycan antibodies are also utilized to diagnose, prognosticate, and monitor disease progression, as well as to study the biological roles and expression of glycans. High-quality anti-glycan mAbs are still in limited supply, highlighting the need for new technologies for anti-glycan antibody discovery. This review discusses anti-glycan monoclonal antibodies with applications to basic research, diagnostics, and therapeutics, focusing on recent advances in mAbs targeting cancer- and infectious disease-associated glycans.     

Point-of-care molecular diagnostic systems--past, present and future

The field of molecular diagnostics has greatly decreased the time it takes to identify infectious agents and to test their antimicrobial resistance. Portable devices are currently in development that can easily identify a variety of nucleic acid targets (either DNA or RNA) from multiple sample types in under an hour. This is done by a variety of methods including real-time polymerase chain reaction, probe-based assays, bioluminescence real-time amplification, and microarray or micro-pump technologies. These self-contained systems require only minimal training and perform all steps of the assay from extraction through detection with little or no operator intervention.     

Existing and Emerging Technologies for Point-of-Care Testing

The volume of point-of-care testing (PoCT) has steadily increased over the 40 or so years since its widespread introduction. That growth is likely to continue, driven by changes in healthcare delivery which are aimed at delivering less costly care closer to the patient’s home. In the developing world there is the challenge of more effective care for infectious diseases and PoCT may play a much greater role here in the future. PoCT technologies can be split into two categories, but in both, testing is generally performed by technologies first devised more than two decades ago. These technologies have undoubtedly been refined and improved to deliver easier-to-use devices with incremental improvements in analytical performance. Of the two major categories the first is small handheld devices, providing qualitative or quantitative determination of an increasing range of analytes. The dominant technologies here are glucose biosensor strips and lateral flow strips using immobilised antibodies to determine a range of parameters including cardiac markers and infectious pathogens. The second category of devices are larger, often bench-top devices which are essentially laboratory instruments which have been reduced in both size and complexity. These include critical care analysers and, more recently, small haematology and immunology analysers. New emerging devices include those that are utilising molecular techniques such as PCR to provide infectious disease testing in a sufficiently small device to be used at the point of care. This area is likely to grow with many devices being developed and likely to reach the commercial market in the next few years.     

Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR

AIMS: Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda-like cheeses. METHODS AND RESULTS: We used ethidium monoazide bromide (EMA)-PCR for direct quantification of viable and dead cells, while semiquantitative detection of culturable cells below the PCR detection limit (c. 100 CFU g(-1)) was obtained by combining growth and real-time PCR. We were able to quantify the fraction of >0.5% viable cells in a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detection limit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitative detection of low levels of culturable cells (10 and 100 CFU g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The significance of this work is that we have developed a novel concept for detection of viable and potentially infectious L. monocytogenes.     

Nanotechnologies for pathogen detection: Future alternatives?

The development of multiplex and flexible tests allowing the simultaneous analysis of pathogens presenting a transfusional risk is a real challenge. Current miniaturized platforms have been particularly marked by microarrays. These microsystems allow the optical detection of hundreds of individual targets simultaneously. However, they suffer from a low sensitivity and their combination with a preliminary target amplification step such as PCR is necessary. The variable level of expression of the infectious genomes of interest and their large diversity complicate multiplex amplification. Finally simultaneous analysis of multiple blood-transmitted agents poses numerous difficulties in diagnosis that remain unresolved by currently available technologies.Until recently, scientific and technological advances for pathogen detection have focused on target amplification and optical detection steps. Today, sample preparation is recognized as a critical area to improve. Nanotechnologies can reach the single-cell or molecular scale and consequently overcome several current technological obstacles. They offer new technological tools for improving sample preparation but also for avoiding target amplification and the current fluorescent labeling. The combination of nano-objects and nano-systems in current technologies offers new possibilities for potential applications in the detection of infectious agents.     

Rapid diagnostic assays in the genomic biology era: detection and identification of infectious disease and biological weapon agents

In this special section of BioTechniques, we examine the role of rapid molecular technologies in the detection and identification of agents of infectious disease (ID) and biological weapons (BWs). Besides the threat posed by the global proliferation of BW technologies, there are numerous emerging and reemerging ID agents with significant public health consequences. Further compounding this already complicated situation are the estimated 600 million international tourists annually, many with the potential to the spread disease globally in a matter of hours. While clinical laboratories have key roles in the detection and identification of potential ID/BW agents, most staff are unfamiliar with these agents because of their rarity and the often laborious conventional methodologies needed to identify them. To meet this challenge, a vast array of rapid assay strategies has been developed for use in clinical diagnostics and environmental detection. Technologies have been developed or adapted to the challenges posed by these agents, permitting detection and identification in several minutes to hours. In particular, the development of improved reagents and detection systems has led to dramatic improvements in the sensitivity and specificity of immunological and nucleic acid-based systems, allowing an ever-increasing range of analytes to be identified and quantitated. In the accompanying articles, we have brought together experts from the many overlapping aspects of this arena in order to present a comprehensive and critical analysis of these technologies.     

Potential applications of DNA microarrays in biodefense-related diagnostics

Recent years have witnessed a logarithmic growth in the number of applications involving DNA microarrays. Extrapolation of their use for infectious diagnostics and biodefense-related diagnostics seems obvious. Nevertheless, the application of DNA microarrays to biodefense-related diagnostics will depend on solving a set of substantial, yet approachable, technical and logistical problems that encompass diverse topics from amplification efficiency to bioinformatics.     

单个B细胞抗体制备技术及应用

单克隆抗体是现代生命科学研究的重要工具,为许多领域的发展作出了不可估量的贡献。随着PCR技术和单克隆抗体技术的发展和成熟,单个B细胞抗体制备技术迅速兴起。该技术能够对单个的抗原特异性B细胞进行抗体基因的体外克隆和表达,保证了轻重链可变区的天然配对,相较于传统的抗体制备技术具有效率高、全人源、基因多样性更丰富等优势。单个B细胞抗体制备技术已成为制备全人抗体的热门方法,同时也促进了包括抗体发生成熟、疫苗保护机制、疫苗开发、肿瘤及自身免疫疾病等免疫学相关研究。文中就单个B细胞抗体制备技术的过程及应用作简要综述。     

基于CRISPR的快速灵敏便捷分子检测

快速灵敏检测技术对疾病防控必不可少。特别是新冠疫情暴发以来,人们深刻认识到快速灵敏检测技术的重要性。近年来,以CRISPR/Cas为代表的基因编辑技术带来了生物技术革命性的进步。CRISPR的核酸检测技术因其快速、准确、灵敏、经济等特点,正在引发分子诊断革新,并已被成功应用于传染病、遗传病、肿瘤基因突变诊断,以及食品安全等领域。本文归纳了基于CRISPR的多种核酸检测体系及应用,并对未来CRISPR核酸检测发展趋势及结合人工智能的智能化检测进行了展望。     

Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples

Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.     

Post-genomic challenges for collaborative research in infectious diseases

Although high-burden pathogens have been prioritized for sequencing, genomic research has yet to yield effective vaccines, diagnostics or therapeutics for the infectious diseases that burden developing countries. International research partnerships are needed more today than ever before, and we propose that increased participation by scientists in endemic areas would overcome current roadblocks and is an essential path towards translational research outcomes.     

Microbe hunting in laboratory animal research

Recent advances in nucleic acid diagnostic technologies have revolutionized microbiology by facilitating rapid, sensitive pathogen surveillance and differential diagnosis of infectious diseases. With the expansion and dissemination of genomic sequencing technology scientists are discovering new microbes at an accelerating pace. In this article we review recent progress in the field of pathogen surveillance and discovery with a specific focus on applications in the field of laboratory animal research. We discuss the challenges in proving a causal relationship between the presence of a candidate organism and disease. We also discuss the strengths and limitations of various assay platforms and describe a staged strategy for viral diagnostics. To illustrate the complexity of pursuing pathogen discovery research, we include examples from our own work that are intended to provide insights into the process that led to the selection of particular strategies.     

Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics.