Advancements in detection of SARS-CoV-2 infection for confronting COVID-19 pandemics.

As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy.

Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review).

Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.

Droplet and Microchamber-Based Digital Loop-Mediated Isothermal Amplification (dLAMP).

Digital loop-mediated isothermal amplification (dLAMP) refers to compartmentalizing nucleic acids and LAMP reagents into a large number of individual partitions, such as microchambers and droplets. This compartmentalization enables dLAMP to be an excellent platform to quantify the absolute number of the target nucleic acids. Owing to its low requirement for instrumentation complexity, high specificity, and strong tolerance to inhibitors in the nucleic acid samples, dLAMP has been recognized as a simple and accurate technique to quantify pathogenic nucleic acid. Herein, the general process of dLAMP techniques is summarized, the current dLAMP techniques are categorized, and a comprehensive discussion on different types of dLAMP techniques is presented. Also, the challenges of the current dLAMP are illustrated together with the possible strategies to address these challenges. In the end, the future directions of the dLAMP developments, including multitarget detection, multisample detection, and processing nucleic acid extraction are outlined. With recently significant advances in dLAMP, this technology has the potential to see more widespread use beyond the laboratory in the future.

Diagnostic and Treatment Strategies for COVID-19.

The world is facing lockdown for the first time in decades due to the novel coronavirus COVID-19 (SARS-CoV-2) pandemic. This has led to massive global economic disruption, placed additional strain on local and global public health resources and, above all, threatened human health. We conducted a review of peer-reviewed and unpublished data, written in English, reporting on the current COVID-19 pandemic. This data includes previously used strategies against infectious disease, recent clinical trials and FDA-approved diagnostic and treatment strategies. The literature was obtained through a systematic search using PubMed, Web of Sciences, and FDA, NIH and WHO websites. Of the 98 references included in the review, the majority focused on pathogen and host targeting, symptomatic treatment and convalescent plasma utilization. Other sources investigated vaccinations in the pipeline for the possible prevention of COVID-19 infection. The results demonstrate various conventional as well as potentially advanced in vitro diagnostic approaches (IVD) for the diagnosis of COVID-19. Mixed results have been observed so far when utilising these approaches for the treatment of COVID-19 infection. Some treatments have been found highly effective in specific regions of the world while others have not altered the disease process. The responsiveness of currently available options is not conclusive. The novelty of this disease, the rapidity of its global outbreak and the unavailability of vaccines have contributed to the global public's fear. It is concluded that the exploration of a range of diagnostic and treatment strategies for the management of COVID-19 is the need of the hour.

Understanding the Promises and Hurdles of Metagenomic Next-Generation Sequencing as a Diagnostic Tool for Infectious Diseases.

Agnostic metagenomic next-generation sequencing (mNGS) has emerged as a promising single, universal pathogen detection method for infectious disease diagnostics. This methodology allows for identification and genomic characterization of bacteria, fungi, parasites, and viruses without the need for a priori knowledge of a specific pathogen directly from clinical specimens. Although there are increasing reports of mNGS successes, several hurdles need to be addressed, such as differentiation of colonization from infection, extraneous sources of nucleic acid, method standardization, and data storage, protection, analysis, and interpretation. As more commercial and clinical microbiology laboratories develop mNGS assays, it is important for treating practitioners to understand both the power and limitations of this method as a diagnostic tool for infectious diseases.

Next-generation sequencing workflows in veterinary infection biology: towards validation and quality assurance.

Recent advancements in DNA sequencing methodologies and sequence data analysis have revolutionised research in many areas of biology and medicine, including veterinary infection biology. New technology is poised to bridge the gap between the research and diagnostic laboratory. This paper defines the potential diagnostic value and purposes of next-generation sequencing (NGS) applications in veterinary infection biology and explores their compatibility with the existing validation principles and methods of the World Organisation for Animal Health. Critical parameters for validation and quality control (quality metrics) are suggested, with reference to established validation and quality assurance guidelines for NGS-based methods of diagnosing human heritable diseases. Although most currently described NGS applications in veterinary infection biology are not primary diagnostic tests that directly result in control measures, this critical reflection on the advantages and remaining challenges of NGS technology should stimulate discussion on its diagnostic value and on the potential to validate NGS methods and monitor their diagnostic performance.

Les avancées récentes enregistrées en matière de séquençage de l'ADN et d'analyse des données de séquences ont révolutionné la recherche dans de nombreux domaines de la biologie et de la médecine, notamment la biologie des maladies animales infectieuses. Ces nouvelles technologies vont permettre de combler le fossé qui séparait la recherche fondamentale du laboratoire de diagnostic. Après avoir défini l'intérêt diagnostique des applications du séquençage de nouvelle génération (SNG) ainsi que leurs finalités dans le domaine de la biologie des maladies animales infectieuses, les auteurs examinent leur compatibilité avec les méthodes et les principes actuels de validation recommandés par l'Organisation mondiale de la santé animale. Ils proposent quelques paramètres critiques de validation et de contrôle qualité (mesure de la qualité), en se référant aux lignes directrices de validation et d'assurance qualité des techniques diagnostiques basées sur le séquençage de nouvelle génération visant à détecter les maladies humaines héréditaires. Certes, la plupart des applications actuelles des méthodes de séquençage de nouvelle génération en biologie des maladies animales infectieuses ne constituent pas des tests de diagnostic primaire (dont dépendent directement les décisions de contrôle sanitaire) ; toutefois, l'analyse critique proposée par les auteurs sur les avantages de cette technologie et sur les difficultés restant à résoudre devrait ouvrir la voie à des discussions sur l'intérêt diagnostique des méthodes recourant au séquençage de nouvelle génération ainsi que sur les perspectives de validation et de contrôle de leurs performances diagnostiques.

Los recientes avances en los métodos de secuenciación del ADN y el análisis de los datos de secuencias han revolucionado la investigación en muchos ámbitos de la biología y la medicina, entre ellos la biología de las infecciones veterinarias. Las nuevas técnicas encierran la promesa de reducir la distancia entre el mundo de la investigación y los laboratorios de diagnóstico. Tras explicar el interés que pueden revestir las aplicaciones de la secuenciación de próxima generación y su posible uso con fines de diagnóstico de infecciones veterinarias, los autores examinan su compatibilidad con los principios y métodos de validación que tiene definidos la Organización Mundial de Sanidad Animal. Asimismo, proponen parámetros básicos para su validación y control de calidad (medición de la calidad), haciendo referencia a las pautas ya establecidas de validación y garantía de calidad de métodos de diagnóstico de enfermedades humanas hereditarias que reposan en técnicas de secuenciación de próxima generación. Aunque la mayoría de las aplicaciones de estas técnicas actualmente descritas en biología de las infecciones veterinarias no constituyen pruebas primarias de diagnóstico, esto es, cuyos resultados puedan inducir directamente medidas de control, esta crucial reflexión sobre las ventajas que entraña la secuenciación de próxima generación y los problemas que aún plantea debería alentar un debate sobre su interés para labores de diagnóstico y sobre la posibilidad de validar métodos basados en estas técnicas y de hacer un seguimiento de la eficacia diagnóstica que ofrezcan.

24-chromosome PCR for aneuploidy screening.

PURPOSE OF REVIEW: The purpose of this study is to describe the many advantages of PCR-based comprehensive chromosome screening (CCS) methodologies and the importance of extensive preclinical validation. RECENT FINDINGS: The rigorous preclinical validation of quantitative real-time (q)PCR-based CCS involved an initial validation on cell lines, followed by a blinded evaluation on embryos. Comparison with alternative platforms and a prospective randomized clinical trial demonstrate superior precision and improved sustained implantation and delivery rates. Preclinical validation of targeted PCR-based next-generation sequencing (NGS) has also demonstrated consistency in positive controls, equivalent accuracy to commonly used techniques, high resolution, increased throughput, the simultaneous detection of single gene disorders and triploidy, and the potential to decrease costs. Prospective randomized controlled trials are ongoing to validate this technique for clinical use. SUMMARY: CCS using PCR-based methodology improves implantation and delivery rates and allows for fresh embryo transfers. Other platforms are available to select euploid embryos; however, there are meaningful differences between techniques. PCR-based NGS technology may further enhance the utility of CCS for patients with infertility.

[Development of molecular detection of food-borne pathogenic bacteria using miniaturized microfluidic devices]. / Élelmiszer-biztonsági jelentoségu baktériumok molekuláris detektálásának fejlesztése miniatürizált mikrofluidikai eszközök segítségével.

Detection and identification of food-borne pathogenic bacteria are key points for the assurance of microbiological food safety. Traditional culture-based methods are more and more replaced by or supplemented with nucleic acid based molecular techniques, targeting specific (preferably virulence) genes in the genomes. Internationally validated DNA amplification - most frequently real-time polymerase chain reaction - methods are applied by the food microbiological testing laboratories for routine analysis, which will result not only in shortening the time for results but they also improve the performance characteristics (e.g. sensitivity, specificity) of the methods. Beside numerous advantages of the polymerase chain reaction based techniques for routine microbiological analysis certain drawbacks have to be mentioned, such as the high cost of the equipment and reagents, as well as the risk of contamination of the laboratory environment by the polymerase chain reaction amplicons, which require construction of an isolated laboratory system. Lab-on-a-chip systems can integrate most of these laboratory processes within a miniaturized device that delivers the same specificity and reliability as the standard protocols. The benefits of miniaturized devices are: simple - often automated - use, small overall size, portability, sterility due to single use possibility. These miniaturized rapid diagnostic tests are being researched and developed at the best research centers around the globe implementing various sample preparation and molecular DNA amplification methods on-chip. In parallel, the aim of the authors' research is to develop microfluidic Lab-on-a-chip devices for the detection and identification of food-borne pathogenic bacteria.

Loop-mediated isothermal amplification (LAMP) assays for detection and identification of aquaculture pathogens: current state and perspectives.

Since its invention in 2000, loop-mediated isothermal amplification (LAMP) assay has been one of the most extensively used molecular diagnostic tools in bio-medical fields due to the rapidity, accuracy, and cost-effectiveness of the technique. This technique has also earned popularity in aquaculture disease diagnosis. Aquaculture, as a result of its rapid intensification and expansion, experiences increased infectious disease occurrences. For maintenance of economic viability, rapid, sensitive and efficient diagnosis of disease causing agents is an important step prior to undertaking effective prevention and control measures in aquaculture. Constraints on time and expertise required for conventional biochemical, serological and polymerase chain reaction (PCR)-based techniques offer avenues in adoption of the LAMP by the aquaculturists at field conditions. This assay has been successfully applied in detection of several bacterial, viral and parasitic pathogens causing serious diseases in aquaculture. In this review, we endeavored to accommodate the LAMP methodology with its different recent improvements and an overview of its application for the detection of aquaculture-associated pathogens.

Loop-mediated isothermal amplification (LAMP): recent progress in research and development.

Loop-mediated isothermal amplification (LAMP) is an established technology that continues to attract the attention of researchers in many fields. Research and development efforts on LAMP technology in recent years have focused on two major areas; first, the study of its clinical application as an approved in vitro diagnostics tool in Japan and certain other countries; and second, research aimed at further simplifying the LAMP test process. This review provides an overview of the status of LAMP on these two topics by summarizing research work conducted, in the main, after our previous review article.

Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern.

With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/µL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.

Recent advances in signal amplification strategy based on oligonucleotide and nanomaterials for microRNA detection-a review.

MicroRNAs (MiRNAs) play multiple crucial regulating roles in cell which can regulate one third of protein-coding genes. MiRNAs participate in the developmental and physiological processes of human body, while their aberrant adjustment will be more likely to trigger diseases such as cancers, kidney disease, central nervous system diseases, cardiovascular diseases, diabetes, viral infections and so on. What's worse, for the detection of miRNAs, their small size, high sequence similarity, low abundance and difficult extraction from cells impose great challenges in the analysis. Hence, it's necessary to fabricate accurate and sensitive biosensing platform for miRNAs detection. Up to now, researchers have developed many signal-amplification strategies for miRNAs detection, including hybridization chain reaction, nuclease amplification, rolling circle amplification, catalyzed hairpin assembly amplification and nanomaterials based amplification. These methods are typical, feasible and frequently used. In this review, we retrospect recent advances in signal amplification strategies for detecting miRNAs and point out the pros and cons of them. Furthermore, further prospects and promising developments of the signal-amplification strategies for detecting miRNAs are proposed.

Sex determination in the wild: a field application of loop-mediated isothermal amplification successfully determines sex across three raptor species.

PCR-based methods are the most common technique for sex determination of birds. Although these methods are fast, easy and accurate, they still require special facilities that preclude their application outdoors. Consequently, there is a time lag between sampling and obtaining results that impedes researchers to take decisions in situ and in real time considering individuals' sex. We present an outdoor technique for sex determination of birds based on the amplification of the duplicated sex-chromosome-specific gene Chromo-Helicase-DNA binding protein using a loop-mediated isothermal amplification (LAMP). We tested our method on Griffon Vulture (Gyps fulvus), Egyptian Vulture (Neophron percnopterus) and Black Kite (Milvus migrans) (family Accipitridae). We introduce the first fieldwork procedure for sex determination of animals in the wild, successfully applied to raptor species of three different subfamilies using the same specific LAMP primers. This molecular technique can be deployed directly in sampling areas because it only needs a voltage inverter to adapt a thermo-block to a car lighter and results can be obtained by the unaided eye based on colour change within the reaction tubes. Primers and reagents are prepared in advance to facilitate their storage at room temperature. We provide detailed guidelines how to implement this procedure, which is simpler (no electrophoresis required), cheaper and faster (results in c. 90 min) than PCR-based laboratory methods. Our successful cross-species application across three different raptor subfamilies posits our set of markers as a promising tool for molecular sexing of other raptor families and our field protocol extensible to all bird species.

Fifteen years of Nucleic Acid Testing in France: Results and lessons.

Of the 40 million donations screened with Nucleic acid testing (NAT) between July 2001 and December 2015 in France, 20 HIV-positive, 13 HCV-positive and 17 HBV (HBV-NAT was initiated in 2005 and extended to the whole country in 2010) donations were discarded thanks to NAT. The main benefit in terms of discarded donations is related to HBV with a yield of 0.88 per million donations, which is 12.5 and 1.8 times higher than for HCV and HIV respectively. The main risk factor found in these donors during the post donation interview was having sex with men for males (n=11, all repeat blood donors), having a partner HCV positive (n=6) or at-risk partner (originated from endemic area or HBV positive) for HBV (n=8) for HIV, HCV and HBV, respectively. Although the mean viral load was high for HIV (5.6 log copies/mL) and HCV (7 log IU/mL), HBV cases show low level of DNA (1.8 log IU/mL) demonstrating the need of a highly sensitive NAT assay. Overall, the clinical benefit for recipients remains those related to the prevention of HIV contaminations since HCV avoided transmissions are extremely rare (only one case in the last 5 years thanks to NAT) and the potential infectivity of HBV-NAT only positive cases is questionable due to the low level of HBV DNA and the presence of anti-HBs in more than a half of DNA positive/HBsAg and anti-HBc negative donors.

Analyzing genes using closing and replicating circles.

During the past two years, significant breakthroughs have been achieved in genetic analyses through the application of technologies based on analytical DNA-circularization reactions. Padlock probes and molecular inversion probes have enabled parallel, high-throughput single nucleotide polymorphism (SNP) genotyping at increased scales, whereas, at the other end of the analysis spectrum, DNA molecules in individual cells have been genotyped, in situ, using padlock probes and rolling-circle amplification (RCA). This review describes the recent developments in the technologies that use specific DNA circularization, coupled to DNA amplification through PCR or rolling-circle amplification, and addresses the great potential of these tools.

Multiple displacement amplification to create a long-lasting source of DNA for genetic studies.

In many situations there may not be sufficient DNA collected from patient or population cohorts to meet the requirements of genome-wide analysis of SNPs, genomic copy number polymorphisms, or acquired copy number alternations. When the amount of available DNA for genotype analysis is limited, high performance whole-genome amplification (WGA) represents a new development in genetic analysis. It is especially useful for analysis of DNA extracted from stored histology slides, tissue samples, buccal swabs, or blood stains collected on filter paper. The multiple displacement amplification (MDA) method, which relies on isothermal amplification using the DNA polymerase of the bacteriophage phi29, is a recently developed technique for high performance WGA. This review addresses new trends in the technical performance of MDA and its applications to genetic analyses. The main challenge of WGA methods is to obtain balanced and faithful replication of all chromosomal regions without the loss of or preferential amplification of any genomic loci or allele. In multiple comparisons to other WGA methods, MDA appears to be most reliable for genotyping, with the most favorable call rates, best genomic coverage, and lowest amplification bias.

Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory.

BACKGROUND: Recent advances in nucleic acid-based methods to detect bacteria offer increased sensitivity and specificity over traditional microbiological techniques. The potential benefit of nucleic acid-based testing to the clinical laboratory is reduced time to diagnosis, high throughput, and accurate and reliable results. METHODS: Several PCR and hybridization tests are commercially available for specific organism detection. Furthermore, hundreds of nucleic acid-based bacterial detection tests have been published in the literature and could be adapted for use in the clinical setting. Contamination potential, lack of standardization or validation for some assays, complex interpretation of results, and increased cost are possible limitations of these tests, however, and must be carefully considered before implementing them in the clinical laboratory. CONCLUSIONS: A major area of advancement in nucleic acid-based assay development has been for specific and broad-range detection of bacterial pathogens.

Present and future of rapid and/or high-throughput methods for nucleic acid testing.

BACKGROUND: Behind the success of 'completing' the human genome project was a more than 30-year history of technical innovations for nucleic acid testing. METHODS: Discovery of specific restriction endonucleases and reverse transcriptase was followed shortly by the development of the first diagnostic nucleic acid tests in the early 1970s. Introduction of Southern, Northern and dot blotting and DNA sequencing later in the 1970s considerably advanced the diagnostic capabilities. Nevertheless, it was the discovery of the polymerase chain reaction (PCR) in 1985 that led to an exponential growth in molecular biology and the introduction of practicable nucleic acid tests in the routine laboratory. The past two decades witnessed a continuing explosion of technological innovations in molecular diagnostics. In addition to classic PCR and reverse transcriptase PCR, numerous variations of PCR and alternative amplification techniques along with an ever-increasing variety of detection chemistries, closed tube (homogeneous) assays, and automated systems were developed. Discovery of real-time quantitative PCR and the development of oligonucleotide microarrays, the 'DNA chip', in the 1990s heralded the beginning of another revolution in molecular biology and diagnostics that is still in progress.

Tracking genes from seed to supermarket: techniques and trends.

Analytical techniques to track plant genes in the environment and the food chain are essential for environmental risk assessment, government regulation and production and trade of genetically modified (GM) crops. Here, I review laboratory techniques to track plant genes during pre-commercialization research on gene flow and post-commercialization detection, identification and quantification of GM crops from seed to supermarket. At present, DNA- and protein-based assays support both activities but the demand for fast, inexpensive, sensitive methods is increasing. Part of the demand has been generated by stringent food labeling and traceability regulations for GM crops. The increase in GM crops, changes in GM crop design, evolution of government regulations and adoption of risk-assessment frameworks will continue to drive development of analytical techniques.