Nucleic Acid Diagnostics Research Articles

Reliable Rapid Assay for Gonorrhea and Chlamydia in the Emergency Department

BackgroundChlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are common sexually transmitted infections seen in the emergency department (ED). Due to an inability to reliably make accurate diagnosis by physical examination, concern for unreliable follow-up, and current delays in diagnostic nucleic acid amplification testing (NAAT), presumptive treatment active against CT and NG, as described by Centers for Disease Control clinical practice guidelines, is often performed. ObjectivesThe purpose of this study was to determine whether a rapid, urine NAAT performed in the ED is noninferior in its diagnostic sensitivity compared with a traditional, swab NAAT assay. MethodsWe performed a prospective, noninferiority study comparing two U.S. Food and Drug Administration-approved NAAT assays for CT and NG: a 90-min rapid assay, the Xpert CT/NG Assay (Cepheid, Sunnyvale, CA) using a urine sample vs. a traditional assay, the Aptima Combo 2 Assay (Gen-Probe Incorporated, San Diego, CA) using a swab sample. This study was registered on Clinicaltrials.gov (NCT02386514). ResultsA total of 1162 patient samples were included in the primary analysis. We observed excellent kappa agreement between assays: NG for men, 1.00 (95% confidence interval [CI] 1.00–1.00); NG for women, 0.87 (95% CI 0.79–0.94); CT for men, 0.81 (95% CI 0.59–1.00); and CT for women: 0.85 (95% CI 0.80–0.90), as well as excellent negative and positive predictive values for the rapid assay. ConclusionAlthough the rapid Xpert CT/NG assay's diagnostic sensitivity did not meet our prespecified threshold for noninferiority, the diagnostic characteristics are robust enough to fit into a management pathway that may reduce unnecessary antibiotic use. There may be an opportunity to utilize the rapid Xpert CT/NG assay to improve accuracy of treatment in the ED.

A technology platform for digital nucleic acid diagnostics at the point of care

Abstract The combination of digital amplification and centrifugal microfluidics can enable quantitative and fast diagnostics at the point of care (PoC). The new unit operation of centrifugal step emulsification allows high throughput droplet generation. Different methods for digital nucleic acid analysis, including PCR, recombinase polymerase amplification (RPA) and loop mediated isothermal amplification (LAMP), have already been demonstrated. Our novel approach of integrated sample-to-answer analysis is introduced, and examples for the detection of HIV and single cell analysis of antibiotic resistant bacteria are presented. Next to these LabDisk based systems, a microfluidic cartridge termed DropChip allows for digital amplification using only commercially available laboratory devices.

Self-powered chip enables nucleic acid diagnostics

The ability of doctors to use nucleic acid-based diagnostic tests in their offices rather than sending out blood samples to contract clinical labs will require simple, easy-to-use devices. Luke P. Lee and coworkers at the University of California, Berkeley, have developed a prototype—a disposable, inexpensive microfluidic device about the size of a microscope slide that only needs a drop of blood and even carries its own power supply (Sci. Adv. 2017, DOI: 10.1126/sciadv.1501645). The researchers pattern the reagents needed for nucleic acid amplification directly into microwells on the chip. When in operation, blood flows through the microfluidic channels, the blood cells are removed, and the plasma is partitioned into the microwells where nucleic acid amplification occurs upon heating in an oven or by reusable heat packs. Sequences of interest are detected by fluorescence readings. The chip is powered by a precharged vacuum battery, which pumps samples by pulling air through

The role of cell-free nucleic acid diagnostics in clinical chemistry and pathology

The role of cell-free nucleic acid diagnostics in clinical chemistry and pathology

Translating Nucleic Acid Amplification Assays to the Microscale: Lab on a Chip for Point-of-Care Molecular Diagnostics

Background: Nucleic acid amplification test (NAAT) provides the highest levels of sensitivity and specificity for sequence-specific detection of DNA and RNA associated with pathogens and diseases. Currently, these tests are largely limited to laboratory use, as they require considerable sample preparation and expensive instrumentation. However, emerging microfluidic lab-on-a-chip technology enables lowcost, easy-to-use, palm-sized NAAT devices, widening NAAT applications to point-of-care (POC) diagnostics. Here, we review the evolution of microfluidic-based molecular diagnostics with a focus on work that has been carried out in our laboratory as an instructive case study. Methods: We discuss technology pathways for integration of pathogen lysis, nucleic acid isolation, amplification, and quantitative detection of DNA and RNA in a microfluidic format suitable for POC diagnostics. In addition, novel microfluidic approaches for plasma extraction, and enzymatic amplification reaction-diffusion-based method (nuclemeter technology) for end-point quantification are described. Results: Simplifications in design and operation of the microfluidic-based molecular (nucleic acid) diagnostics can be realized with modifications to solid-phase extraction methods to isolate, purify, and concentrate nucleic acid; the use of isothermal amplification; pre-loaded, paraffin-encapsulated reagents; chemical heating; and Smartphone-based detection. Conclusion: The future development of microfluidic-based POC devices is to fully integrate multiple processes required for genetic analysis on a single microfluidic format platform. There is an urgent need for such inexpensive, POC, molecular diagnostic systems at the bedside, at home, in doctors’ and dentists’ offices, clinics, and pharmacies, and especially in resource-limited regions of the world. Keywords: Lab-on-a-chip, microfluidics, molecular diagnostics, nucleic acid amplification test (NAAT), point-of-care (POC).

Adaptation and Validation of E-Probe Diagnostic Nucleic Acid Analysis for Detection of Escherichia coli O157:H7 in Metagenomic Data from Complex Food Matrices

The Centers for Disease Control and Prevention recently emphasized the need for enhanced technologies to use in investigations of outbreaks of foodborne illnesses. To address this need, e-probe diagnostic nucleic acid analysis (EDNA) was adapted and validated as a tool for the rapid, effective identification and characterization of multiple pathogens in a food matrix. In EDNA, unassembled next generation sequencing data sets from food sample metagenomes are queried using pathogen-specific sequences known as electronic probes (e-probes). In this study, the query of mock sequence databases demonstrated the potential of EDNA for the detection of foodborne pathogens. The method was then validated using next generation sequencing data sets created by sequencing the metagenome of alfalfa sprouts inoculated with Escherichia coli O157:H7. Nonspecific hits in the negative control sample indicated the need for additional filtration of the e-probes to enhance specificity. There was no significant difference in the ability of an e-probe to detect the target pathogen based upon the length of the probe set oligonucleotides. The results from the queries of the sample database using E. coli e-probe sets were significantly different from those obtained using random decoy probe sets and exhibited 100% precision. The results support the use of EDNA as a rapid response methodology in foodborne outbreaks and investigations for establishing comprehensive microbial profiles of complex food samples.

Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes.

The development of molecular strategies that enable recognition of specific double-stranded DNA (dsDNA) regions has been a longstanding goal as evidenced by the emergence of triplex-forming oligonucleotides, peptide nucleic acids (PNAs), minor groove binding polyamides, and—more recently—engineered proteins such as CRISPR/Cas9. Despite this progress, an unmet need remains for simple hybridization-based probes that recognize specific mixed-sequence dsDNA regions under physiological conditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probes are chimeras between pseudocomplementary DNA (pcDNA) and Invader probes, which are activated for mixed-sequence dsDNA-recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalized nucleotides, respectively. We demonstrate that certain pseudocomplementary Invader probe designs result in very efficient and specific recognition of model dsDNA targets in buffers of high ionic strength. These chimeric probes, therefore, present themselves as a promising strategy for mixed-sequence recognition of dsDNA targets for applications in molecular biology and nucleic acid diagnostics.

The Presentation, Diagnosis, and Treatment of Sexually Transmitted Infections.

The reported incidence of sexually transmitted infections (STIs) in Germany is rising. For example, the number of new reported cases of syphilis rose from 3034 in 2010 to 4410 in 2012. This review is based on pertinent articles retrieved by a selective search in MEDLINE, and on guidelines and systematic reviews from Germany and abroad. We discuss sexually transmitted infections presenting with genital, anal, perianal, or oral ulcers, urethritis, cervicitis, urethral or vaginal discharge, or genital warts. We also discuss sexually transmitted infection with HIV and the hepatitis C virus (HCV). Acquired sexually transmitted infections elevate the risk of transmission of other sexually transmitted infections; thus, patients presenting for the diagnosis or treatment of any kind of sexually transmitted infection should be evaluated for others as well. For most of these diseases, treatment of the patient's sexual partner(s) is indicated. Diagnostic nucleic acid amplification techniques are over 90% sensitive and specific and are generally the best way to detect the responsible pathogen. Factors impeding effective treatment include antibiotic resistance (an increasing problem) and the late diagnosis of HIV and HCV infections. Sexually transmitted infections are common around the world, and any such infection increases the patient's risk of contracting other types of sexually transmitted infection. Molecular genetic diagnostic techniques should be made widely available.

Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip.

A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon.

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

Detection of low-abundance nucleic acids is a challenging task, which over the last two decades has been solved using enzymatic target amplification. Enzymatic synthesis enhances the signal so that diverse, scientifically and clinically relevant molecules can be identified and studied, including cancer DNA, viral nucleic acids, and regulatory RNAs. However, using enzymes increases the detection time and cost, not to mention the high risk of mistakes with amplification and data alignment. These limitations have stimulated a growing interest in enzyme-free methods within researchers and industry. In this review we discuss recent advances in signal-enhancing approaches aimed at nucleic acid diagnostics that do not require target amplification. Regardless of enzyme usage, signal enhancement is crucial for the reliable detection of nucleic acids at low concentrations. We pay special attention to novel nanomaterials, fluorescence microscopy, and technical advances in detectors for optical assessment. We summarize sensitivity parameters of the currently available assays and devices which makes this review relevant to the broad spectrum of researchers working in fields from biophysics, to engineering, to synthetic biology and bioorganic chemistry.

Progress in multiplex loop-mediated isothermal amplification technology.

Loop-mediated isothermal amplification (LAMP) has been widely applied in nucleic acid diagnostics due to its high sensitivity and specificity, high speed and low requirement of equipment. In order to fully leverage these merits, achieve high efficiency and reliability in diagnostics, and expand the applicable fields while keeping low reagent cost, multiplex LAMP technology has been extensively explored in recent years. Common methods for LAMP products detection are mostly based on the double-stranded DNA amplicons or byproducts from the polymerization reaction, so they can only identify the occurrence of amplification reaction but not the origins or specificity of the products. To achieve specific LAMP products detection, researchers developed various multiplex methods by improving the conventional LAMP technology or coupling LAMP with other assays. However, the interference and/or the different amplification efficiencies among different primer sets often lead to biased amplification and thus limited multiplexing level. We here defined these methods as narrow-sensed multiplex LAMP. The research on miniaturized amplification technology which is booming in recent years has given rise to the novel general-sensed multiplex LAMP technology that breaks this limitation by its capability to perform highly parallel and miniaturized simplex reactions in independent compartments. Methods of this type have additional benefits such as lower reagent cost, higher level of automation, lower risk of cross-contamination and better suitability for on-site detection of multiple targets. In this review, we summarize the recent research progress in multiplex LAMP technology from the following aspects: the principle and design of narrow-sensed LAMP and its amplification optimization, the general-sensed LAMP, and the various applications of all multiplex LAMP technologies in diagnostics.

Multiplex detection and genotyping of pathogenic bacteria on paper-based biosensor with a novel universal primer mediated asymmetric PCR.

Traditionary multiplex asymmetric polymerase chain reaction (PCR) can be applied to detect multiplex target organisms simultaneously, but complex optimizations of primer concentrations and staggered additions of primers are required to achieve equal amplification of multiplex genes. To overcome this shortcoming, we propose a novel method based on multiplex asymmetric PCR and paper-based nucleic acid diagnostics (PBNAD). In the asymmetric PCR, a universal primer was introduced to break the bottlenecks of low sensitivity and self-inhibition among different sets of primers. Amplification using the novel multiplex asymmetric PCR boosted the quantity of single-stranded amplicons, and the amplified products contained the same sequence at the 5' end. Therefore, only one gold nanoparticle-based signal probe was needed for the simultaneous detection of three genes using the PBNAD platform, and the detection signals could be observed with the naked eye. With this highly efficient, novel multiplex asymmetric PCR, as little as 1 pg/μL genomic DNA can be detected. This method can also be applied to genotyping for reliable epidemiological investigations. This proof-of-concept study highlights the potential of the PBNAD platform for cost- and labor-effective applications in the detection of pathogenic bacteria.

Detecting DNA viruses in oral fluids: evaluation of collection and storage methods

Storing saliva for nucleic acid diagnostics is problematic in resource-constrained settings. DNA Genotek's OMNIgene™·DISCOVER kit aims to stabilise microbial DNA at room temperature. We evaluate this for long-term storage, determining DNA quantity/purity and human herpesvirus 8 (HHV-8) load as indicator. Viral loads and DNA degradation were assayed over 14months in HHV-8–negative saliva spiked with cell-associated and cell-free virus and saliva collected fresh frozen and into kits from 10 HIV-positive patients. Viral loads remained constant for 6–9months, yielding high quantities of DNA: subsequent losses were ≤48%. Patient samples, frozen or kit stored, produced pure DNA of comparable concentration. Higher HHV-8 detection in frozen saliva resulted from losses during ethanol precipitation using kits. After 14months, DNA degradation was significant in frozen saliva, but that in kits had integrity similar to fresh samples. Storing frozen saliva is detrimental. This kit is well suited for collection, long-term storage, and assay of viral DNA in resource-constrained settings.

A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water.

BackgroundWater and High Purity Water (HPW) distribution systems can be contaminated with human pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid culture independent methodology to quantitatively detect and identify biocontamination associated with HPW.ResultsA novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, to rapidly detect, identify and quantify the human pathogenic bacteria Stenotrophomonas maltophilia, Burkholderia species, Pseudomonas aeruginosa and Serratia marcescens which are commonly associated with the biocontamination of water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P. aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively.Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background microbiological contamination observed, both S. maltophilia and Burkholderia species were quantitatively detected and identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the threshold of 10 cfu/100 ml recommended by both EU and US guidelines.ConclusionsThe novel culture independent methodology described in this study allows for rapid (<5 h), quantitative detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems. We propose that the described NAD assay and associated methodology could be applied to routine testing of water and HPW distribution systems to assure microbiological safety and high water quality standards.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0124-1) contains supplementary material, which is available to authorized users.

Plasmonics-based SERS nanobiosensor for homogeneous nucleic acid detection

Developing a simple and efficient nucleic acid detection technology is essential for clinical diagnostics. Here, we describe a new conceptually simple and selective “turn on” plasmonics-based nanobiosensor, which integrates non-enzymatic DNA strand-displacement hybridization for specific nucleic acid target identification with surface-enhanced Raman scattering (SERS) detection. This SERS nanobiosensor is a target label-free, and rapid nanoparticle-based biosensing system using a homogeneous assay format that offers a simple and efficient tool for nucleic acid diagnostics. Our results showed that the nanobiosensor provided a limit of detection of ~0.1nM (200amol) in the current bioassay system, and exhibited high specificity for single nucleotide mismatch discrimination. From the Clinical EditorSurface-enhanced Raman scattering (SERS) is a sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement means that the technique may even detect single molecules. In this article, the authors describe a simple and efficient nucleic acid detection technology using SERS, with "OFF-to-ON" signal switch upon nucleic acid target identification and capture, which provides high sensitivity and specificity for single nucleotide mismatch discrimination. This new technology will be most welcomed in clinical diagnostics.

Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine.

Conventional anticancer therapies, such as chemo- and/or radio-therapy are often unable to completely eradicate cancers due to abnormal tumor microenvironment, as well as increased drug/radiation resistance. More effective therapeutic strategies for overcoming these obstacles are urgently in demand. Aptamers, as chemical antibodies that bind to targets with high affinity and specificity, are a promising new and novel agent for both cancer diagnostic and therapeutic applications. Aptamer-based cancer cell targeting facilitates the development of active targeting in which aptamer-mediated drug delivery could provide promising anticancer outcomes. This review is to update the current progress of aptamer-based cancer diagnosis and aptamer-mediated active targeting for cancer therapy in vivo, exploring the potential of this novel form of targeted cancer therapy.

A new approach for detecting fungal and oomycete plant pathogens in next generation sequencing metagenome data utilising electronic probes.

Early stage infections caused by fungal/oomycete spores may not be detected until signs or symptoms develop. Serological and molecular techniques are currently used for detecting these pathogens. Next-generation sequencing (NGS) has potential as a diagnostic tool, due to the capacity to target multiple unique signature loci of pathogens in an infected plant metagenome. NGS has significant potential for diagnosis of important eukaryotic plant pathogens. However, the assembly and analysis of huge amounts of sequence is laborious, time consuming, and not necessary for diagnostic purposes. Previous work demonstrated that a bioinformatic tool termed Electronic probe Diagnostic Nucleic acid Analysis (EDNA) had potential for greatly simplifying detecting fungal and oomycete plant pathogens in simulated metagenomes. The initial study demonstrated limitations for detection accuracy related to the analysis of matches between queries and metagenome reads. This study is a modification of EDNA demonstrating a better accuracy for detecting fungal and oomycete plant pathogens.

Nucleic Acid-based approaches for detection of viral hepatitis.

Context:To determining suitable nucleic acid diagnostics for individual viral hepatitis agent, an extensive search using related keywords was done in major medical library and data were collected, categorized, and summarized in different sections.Results:Various types of molecular biology tools can be used to detect and quantify viral genomic elements and analyze the sequences. These molecular assays are proper technologies for rapidly detecting viral agents with high accuracy, high sensitivity, and high specificity. Nonetheless, the application of each diagnostic method is completely dependent on viral agent.Conclusions:Despite rapidity, automation, accuracy, cost-effectiveness, high sensitivity, and high specificity of molecular techniques, each type of molecular technology has its own advantages and disadvantages.

Screening metagenomic data for viruses using the e-probe diagnostic nucleic acid assay.

Next generation sequencing (NGS) is not used commonly in diagnostics, in part due to the large amount of time and computational power needed to identify the taxonomic origin of each sequence in a NGS data set. By using the unassembled NGS data sets as the target for searches, pathogen-specific sequences, termed e-probes, could be used as queries to enable detection of specific viruses or organisms in plant sample metagenomes. This method, designated e-probe diagnostic nucleic acid assay, first tested with mock sequence databases, was tested with NGS data sets generated from plants infected with a DNA (Bean golden yellow mosaic virus, BGYMV) or an RNA (Plum pox virus, PPV) virus. In addition, the ability to detect and differentiate among strains of a single virus species, PPV, was examined by using probe sets that were specific to strains. The use of probe sets for multiple viruses determined that one sample was dually infected with BGYMV and Bean golden mosaic virus.

Metagenomic search strategies for interactions among plants and multiple microbes.

Plants harbor multiple microbes. Metagenomics can facilitate understanding of the significance, for the plant, of the microbes, and of the interactions among them. However, current approaches to metagenomic analysis of plants are computationally time consuming. Efforts to speed the discovery process include improvement of computational speed, condensing the sequencing reads into smaller datasets before BLAST searches, simplifying the target database of BLAST searches, and flipping the roles of metagenomic and reference datasets. The latter is exemplified by the e-probe diagnostic nucleic acid analysis approach originally devised for improving analysis during plant quarantine.