Explore the vast range of titles available.

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR–Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.Specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples.

The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health. This Review summarizes the rapidly expanding toolbox of assays for CRISPR-based diagnostics and highlights their point-of-care applications.

Abstract Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.

The human placenta is a dynamic and heterogeneous organ critical in the establishment of the fetomaternal interface and the maintenance of gestational well-being. It is also the major source of cell-free fetal nucleic acids in the maternal circulation. Placental dysfunction contributes to significant complications, such as preeclampsia, a potentially lethal hypertensive disorder during pregnancy. Previous studies have identified significant changes in the expression profiles of preeclamptic placentas using whole-tissue analysis. Moreover, studies have shown increased levels of targeted RNA transcripts, overall and placental contributions in maternal cell-free nucleic acids during pregnancy progression and gestational complications, but it remains infeasible to noninvasively delineate placental cellular dynamics and dysfunction at the cellular level using maternal cell-free nucleic acid analysis. In this study, we addressed this issue by first dissecting the cellular heterogeneity of the human placenta and defined individual cell-type–specific gene signatures by analyzing more than 24,000 nonmarker selected cells from full-term and early preeclamptic placentas using large-scale microfluidic single-cell transcriptomic technology. Our dataset identified diverse cellular subtypes in the human placenta and enabled reconstruction of the trophoblast differentiation trajectory. Through integrative analysis with maternal plasma cell-free RNA, we resolved the longitudinal cellular dynamics of hematopoietic and placental cells in pregnancy progression. Furthermore, we were able to noninvasively uncover the cellular dysfunction of extravillous trophoblasts in early preeclamptic placentas. Our work showed the potential of integrating transcriptomic information derived from single cells into the interpretation of cell-free plasma RNA, enabling the noninvasive elucidation of cellular dynamics in complex pathological conditions.

The BioWatch program, funded and overseen by the Department of Homeland Security (DHS), has three main elements-sampling, analysis, and response-each coordinated by different agencies. The Environmental Protection Agency maintains the sampling component, the sensors that collect airborne particles. The Centers for Disease Control and Prevention coordinates analysis and laboratory testing of the samples, though testing is actually carried out in state and local public health laboratories. Local jurisdictions are responsible for the public health response to positive findings. The Federal Bureau of Investigation is designated as the lead agency for the law enforcement response if a bioterrorism event is detected. In 2003 DHS deployed the first generation of BioWatch air samplers. The current version of this technology, referred to as Generation 2.0, requires daily manual collection and testing of air filters from each monitor. DHS has also considered newer automated technologies (Generation 2.5 and Generation 3.0) which have the potential to produce results more quickly, at a lower cost, and for a greater number of threat agents. Technologies to Enable Autonomous Detection for BioWatch is the summary of a workshop hosted jointly by the Institute of Medicine and the National Research Council in June 2013 to explore alternative cost-effective systems that would meet the requirements for a BioWatch Generation 3.0 autonomous detection system, or autonomous detector, for aerosolized agents . The workshop discussions and presentations focused on examination of the use of four classes of technologies-nucleic acid signatures, protein signatures, genomic sequencing, and mass spectrometry-that could reach Technology Readiness Level (TRL) 6-plus in which the technology has been validated and is ready to be tested in a relevant environment over three different tiers of temporal timeframes: those technologies that could be TRL 6-plus ready as part of an integrated system by 2016, those that are likely to be ready in the period 2016 to 2020, and those are not likely to be ready until after 2020. Technologies to Enable Autonomous Detection for BioWatch discusses the history of the BioWatch program, the role of public health officials and laboratorians in the interpretation of BioWatch data and the information that is needed from a system for effective decision making, and the current state of the art of four families of technology for the BioWatch program. This report explores how the technologies discussed might be strategically combined or deployed to optimize their contributions to an effective environmental detection capability.

Pooling designs have been widely used in various aspects of DNA sequencing. In biological applications, the well-studied mathematical problem called “group testing” shifts its focus to nonadaptive algorithms while the focus of traditional group testing is on sequential algorithms. Biological applications also bring forth new models not previously considered, such as the error-tolerant model, the complex model, and the inhibitor model. This book is the first attempt to collect all the significant research on pooling designs in one convenient place.

Nucleic acid is the main material for storing, copying, and transmitting genetic information. Gene sequencing is of great significance in DNA damage research, gene therapy, mutation analysis, bacterial infection, drug development, and clinical diagnosis. Gene detection has a wide range of applications, such as environmental, biomedical, pharmaceutical, agriculture and forensic medicine to name a few. Compared with Sanger sequencing, high-throughput sequencing technology has the advantages of larger output, high resolution, and low cost which greatly promotes the application of sequencing technology in life science research. Magnetic nanoparticles, as an important part of nanomaterials, have been widely used in various applications because of their good dispersion, high surface area, low cost, easy separation in buffer systems and signal detection. Based on the above, the application of magnetic nanoparticles in nucleic acid detection was reviewed.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA.

Cell-free DNA (cfDNA) analysis represents a promising method for the diagnosis, treatment selection and clinical follow-up of cancer patients. Although its general methodological feasibility and usefulness has been demonstrated, several issues related to standardisation and technical validation must be addressed for its routine clinical application in cancer. In this regard, most cfDNA clinical applications are still limited to clinical trials, proving its value in several settings. In this paper, we review the current clinical trials involving cfDNA/ctDNA analysis and highlight those where it has been useful for patient stratification, treatment follow-up or development of novel approaches for early diagnosis. Our query included clinical trials, including the terms ‘cfDNA’, ‘ctDNA’, ‘liquid biopsy’ AND ‘cancer OR neoplasm’ in the FDA and EMA public databases. We identified 1370 clinical trials (FDA = 1129, EMA = 241) involving liquid-biopsy analysis in cancer. These clinical trials show promising results for the early detection of cancer and confirm cfDNA as a tool for real-time monitoring of acquired therapy resistance, accurate disease-progression surveillance and improvement of treatment, situations that result in a better quality of life and extended overall survival for cancer patients.