Comparison of the performance of two targeted metagenomic virus capture probe-based methods using reference control materials and clinical samples.

Viral enrichment by probe hybridization has been reported to significantly increase the sensitivity of viral metagenomics. This study compares the analytical performance of two targeted metagenomic virus capture probe-based methods: (i) SeqCap EZ HyperCap by Roche (ViroCap) and (ii) Twist Comprehensive Viral Research Panel workflow, for diagnostic use. Sensitivity, specificity, and limit of detection were analyzed using 25 synthetic viral sequences spiked in increasing proportions of human background DNA, eight clinical samples, and American Type Culture Collection (ATCC) Virome Virus Mix. Sensitivity and specificity were 95% and higher for both methods using the synthetic and reference controls as gold standard. Combining thresholds for viral sequence read counts and genome coverage [respectively 500 reads per million (RPM) and 10% coverage] resulted in optimal prediction of true positive results. Limits of detection were approximately 50-500 copies/mL for both methods as determined by ddPCR. Increasing proportions of spike-in cell-free human background sequences up to 99.999% (50 ng/mL) did not negatively affect viral detection, suggesting effective capture of viral sequences. These data show analytical performances in ranges applicable to clinical samples, for both probe hybridization metagenomic approaches. This study supports further steps toward more widespread use of viral metagenomics for pathogen detection, in clinical and surveillance settings using low biomass samples. IMPORTANCE: Viral metagenomics has been gradually applied for broad-spectrum pathogen detection of infectious diseases, surveillance of emerging diseases, and pathogen discovery. Viral enrichment by probe hybridization methods has been reported to significantly increase the sensitivity of viral metagenomics. During the past years, a specific hybridization panel distributed by Roche has been adopted in a broad range of different clinical and zoonotic settings. Recently, Twist Bioscience has released a new hybridization panel targeting human and animal viruses. This is the first report comparing the performance of viral metagenomic hybridization panels.

Ultra-stable threose nucleic acid-based biosensors for rapid and sensitive nucleic acid detection and in vivo imaging.

The human genome's nucleotide sequence variation, such as single nucleotide mutations, can cause numerous genetic diseases. However, detecting nucleic acids accurately and rapidly in complex biological samples remains a major challenge. While natural deoxyribonucleic acid (DNA) has been used as biorecognition probes, it has limitations like poor specificity, reproducibility, nuclease-induced enzymatic degradation, and reduced bioactivity on solid surfaces. To address these issues, we introduce a stable and reliable biosensor called graphene oxide (GO)- threose nucleic acid (TNA). It comprises chemically modified TNA capture probes on GO for detecting and imaging target nucleic acids in vitro and in vivo, distinguishing single nucleobase mismatches, and monitoring dynamic changes in target microRNA (miRNA). By loading TNA capture probes onto the GO substrate, the GO-TNA sensing platform for nucleic acid detection demonstrates a significant 88-fold improvement in the detection limit compared to TNA probes alone. This platform offers a straightforward preparation method without the need for costly and labor-intensive isolation procedures or complex chemical reactions, enabling real-time analysis. The stable TNA-based GO sensing nanoplatform holds promise for disease diagnosis, enabling rapid and accurate detection and imaging of various disease-related nucleic acid molecules at the in vivo level. STATEMENT OF SIGNIFICANCE: The study's significance lies in the development of the GO-TNA biosensor, which addresses limitations in nucleic acid detection. By utilizing chemically modified nucleic acid analogues, the biosensor offers improved reliability and specificity, distinguishing single nucleobase mismatches and avoiding false signals. Additionally, its ability to detect and image target nucleic acids in vivo facilitates studying disease mechanisms. The simplified preparation process enhances practicality and accessibility, enabling real-time analysis. The biosensor's potential applications extend beyond healthcare, contributing to environmental analysis and food safety. Overall, this study's findings have substantial implications for disease diagnosis, biomedical research, and diverse applications, advancing nucleic acid detection and its impact on various fields.

Stretching the Bisalkyne Raman Spectral Palette Reveals a New Electrophilic Covalent Motif.

Small heteroaryl-diyne (Het-DY) tags with distinct vibrational frequencies, and physiologically relevant cLog P were designed for multiplexed bioorthogonal Raman imaging. Pd-Cu catalyzed coupling, combined with the use of Lei ligand, was shown to improve overall yields of the desired heterocoupled Het-DY tags, minimizing the production of homocoupled side-products. Spectral data were in agreement with the trends predicted by DFT calculations and systematic introduction of electron- rich/poor rings stretched the frequency limit of aryl-capped diynes (2209-2243 cm-1 ). The improved Log P of these Het-DY tags was evident from their diffuse distribution in cellular uptake studies and functionalizing tags with organelle markers allowed the acquisition of location-specific biological images. LC-MS- and NMR-based assays showed that some heteroaryl-capped internal alkynes are potential nucleophile traps with structure-dependent reactivity. These biocompatible Het-DY tags, equipped with covalent reactivity, open up new avenues for Raman bioorthogonal imaging.

Aptasensors versus immunosensors-Which will prevail?

Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element.

Ultrasensitive detection of plant hormone abscisic acid-based surface-enhanced Raman spectroscopy aptamer sensor.

Abscisic acid (ABA), as the most common plant hormone in the growth of wheat, can greatly affect the yield when its levels deviate from normal. Therefore, highly sensitive and selective detection of this hormone is greatly needed. In this work, we developed an aptamer sensor based on surface-enhanced Raman spectroscopy (SERS) and applied it for the high sensitivity detection of ABA. Biotin-modified ABA aptamer complement chains were modified on ferrosoferric oxide magnetic nanoparticles (Fe3O4MNPs) and acted as capture probes, and sulfhydryl aptamer (SH-Apt)-modified silver-coated gold nanospheres (Au@Ag NPs) were used as signal probes. Through the recognition of the ABA aptamer and its complementary chains, an aptamer sensor based on SERS was constructed. As SERS internal standard molecules of 4-mercaptobenzoic acid (4-MBA) were encapsulated between the gold core and silver shell of the signal probes; the constructed aptamer sensor generated a strong SERS signal of 4-MBA after magnetic separation. When there were ABA molecules in the detection system, with the preferential binding of ABA aptamer and ABA molecule, the signal probes were released from the capture probes, after magnetic separation, leading to a linear decrease in SERS intensity of 4-MBA. Thus, the detection response was linear over a logarithmic concentration range, with an ultra-low detection limit of 0.67 fM. In addition, the practical use of this assay method was demonstrated in ABA detection from fresh wheat leaves, with a relative error (RE) of 5.43-8.94% when compared with results from enzyme-linked immunosorbent assay (ELISA). The low RE value proves that the aptamer sensor will be a promising method for ABA detection.

A dual-recognition-controlled electrochemical biosensor for accurate and sensitive detection of specific circulating tumor cells.

Accurate and sensitive assay of specific circulating tumor cells (CTCs) is of importance for the diagnosis, treatment, and metastasis monitoring of cancer. Herein, we have proposed a dual-recognition-controlled electrochemical biosensor in this work for the detection of specific CTCs. To this sensor, two aptamer hairpin probes are designed to be able to separately bind to two adjacent proteins on the cell membrane to activate the associative toehold for strand displacement reaction, which will then trigger a dimer-like rolling cycle amplification reaction, and finally produce significantly amplified electrochemical signals for sensitive quantification of target CTCs. In our design, only the case that the two proteins are simultaneously expressed on the cell membrane can result in obvious signal responses, which may greatly improve the accuracy of CTCs analysis. The proposed biosensor can possess excellent selectivity to distinguish target cells from different cancer cells. Moreover, the combination of rolling cycle amplification and DNA nanostructure capture probes can effectively lower the detection limit to 3 cells mL-1. Notably, our biosensor can be applied to the assay of the target CTCs in the complex whole blood matrixes, verifying its strong stability and anti-interference. Thus, the as-proposed dual-recognition-controlled electrochemical biosensor may exhibit great promise in clinical cancer diagnosis and personalized medicine.

Viral metagenomic sequencing in a cohort of international travellers returning with febrile illness.

BACKGROUND: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyze the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness. METHODS: Thirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods. RESULTS: In 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected. CONCLUSIONS: Viral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing.

Improved diagnosis of viral encephalitis in adult and pediatric hematological patients using viral metagenomics.

Metagenomic sequencing is a powerful technique that enables detection of the full spectrum of pathogens present in any specimen in a single test. Hence, metagenomics is increasingly being applied for detection of viruses in clinical cases with suspected infections of unknown etiology and a large number of relevant potential causes. This is typically the case in patients presenting with encephalitis, in particular when immunity is impaired by underlying disorders. In this study, viral metagenomics has been applied to a cohort of hematological patients with encephalitis of unknown origin. Because viral loads in cerebrospinal fluid of patients with encephalitis are generally low, the technical performance of a metagenomic sequencing protocol with viral enrichment by capture probes targeting all known vertebrate viral sequences was studied. Subsequently, the optimized viral metagenomics protocol was applied to a cohort of hematological patients with encephalitis of unknown origin. Viral enrichment by capture probes increased the viral sequence read count of metagenomics on cerebrospinal fluid samples 100 - 10.000 fold, compared to unenriched metagenomic sequencing. In five out of 41 (12%) hematological patients with encephalitis, a virus was detected by viral metagenomics which had not been detected by current routine diagnostics. BK polyomavirus, hepatitis E virus, human herpes virus-6 and Epstein Barr virus were identified by this unbiased metagenomic approach. This study demonstrated that hematological patients with encephalitis of unknown origin may benefit from early viral metagenomics testing as a single step approach.

Bridging Fe3O4@Au nanoflowers and Au@Ag nanospheres with aptamer for ultrasensitive SERS detection of aflatoxin B1.

Aflatoxin B1 (AFB1) as the most toxic mycotoxin in contaminated food can greatly threaten human health, and sensitive and selective detection of AFB1 is thus highly desired. An ultrasensitive surface-enhanced Raman spectroscopy (SERS) aptasensor was developed for AFB1 detection in peanut oil samples. SH-cDNA modified Fe3O4@Au nanoflowers acted as capture probes, SH-Apt modified Au@Ag nanospheres and commercial Cy3-Apt were used as reporter probes. Strong SERS signals of reporter probes were produced due to the recognition of AFB1 aptamer and its complementary strand (SH-cDNA). With the preferred binding of AFB1 aptamer to AFB1, reporter probes were released from capture probes, causing a linear decrease in SERS intensity. Therefore an ultralow detection limit of 0.40 pg·mL-1 in a wide linear range of 0.0001-100 ng·mL-1 was obtained and the sensibility of this SERS aptasensor was higher than that of the Cy3-Apt based SERS aptasensor. In addition, an excellent selectivity in interfering toxins and satisfactory recoveries of 96.6-115% in peanut oil samples were obtained, proving this aptasensor is a promising analytical tool in AFB1 detection.

CaptureProbe: a java tool for designing probes for capture Hi-C applications.

Many functional elements associated with traits and diseases are located in non-coding regions and act on distant target genes via chromatin looping and folding, making it difficult for scientists to reveal the genetic regulatory mechanisms. Capture Hi-C is a newly developed chromosome conformation capture technology based on hybridization capture between probes and target genomic regions. It can identify interactions among target loci and all other loci in a genome with low cost and high resolution. Here, we developed CaptureProbe, a user-friendly, graphical Java tool for the design of capture probes across a range of target sites or regions. Numerous parameters helped to achieve and optimize the designed probes. Design testing of CaptureProbe showed high efficiency in the design success ratio of target loci and probe specificity. Hence, this program will help scientists conduct genome spatial interaction research. CaptureProbe and source code are available at https://sourceforge.net/projects/captureprobe/.

A Rapid Method for Detection of Salmonella in Milk Based on Extraction of mRNA Using Magnetic Capture Probes and RT-qPCR.

Magnetic separation is an efficient method for target enrichment and elimination of inhibitors in the molecular detection systems for foodborne pathogens. In this study, we prepared magnetic capture probes by modifying oligonucleotides complementary to target sequences on the surface of amino-modified silica-coated magnetic nanoparticles and optimized the conditions and parameters of probe synthesis and hybridization. We innovatively put the complexes of magnetic capture probes and target sequences into qPCR without any need for denaturation and purification steps. This strategy can reduce manual steps and save time. We used the magnetic capture probes to separate invA mRNA from Salmonella in artificially contaminated milk samples. The detection sensitivity was 104 CFU/ml, which could be increased to 10 CFU/ml after a 12 h enrichment step. The developed method is robust enough to detect live bacteria in a complex environmental matrix.

SureSelectXT RNA Direct: A Technique for Expression Analysis Through Sequencing of Target-Enriched FFPE Total RNA.

Gene expression profiling of samples from biobanks requires a method that can be used with intact as well as partially degraded RNA. High throughput applications can benefit from reducing the number of processing steps including eliminating the poly(A) selection and ribosomal depletion steps. When performing targeted capture, we have found that we can eliminate the upfront poly(A) selection/ribosomal depletion steps that cause bias in standard mRNA-Seq workflows. This target enrichment solution allows for whole transcriptome or customized content to characterize differential gene expression patterns (especially for mid/low level transcripts). Protocol modifications to the Agilent Strand-Specific RNA Library Prep kit resulted in a new workflow called "RNA Direct" that generates RNA-Seq data with minimal ribosomal contamination and good sequencing coverage. Using RNA isolated from a set of matched samples including fresh frozen (FF) or formalin-fixed, paraffin-embedded (FFPE) from tumor/normal tissues we generated high-quality data using a protocol that does not require upfront ribosomal depletion or poly(A) selection. Using SureSelectXT RNA Direct protocol (RNA Direct) workflow, we found transcripts to be upregulated or downregulated to similar degrees with similar confidence levels in both the FF and FFPE samples, demonstrating the utility for meaningful gene expression studies with biobank samples of variable quality.

Model selection as a tool for phylogeographic inference: an example from the willow Salix melanopsis.

Phylogeographic inference has typically relied on analyses of data from one or a few genes to provide estimates of demography and population histories. While much has been learned from these studies, all phylogeographic analysis is conditioned on the data, and thus, inferences derived from data that represent a small sample of the genome are unavoidably tenuous. Here, we demonstrate one approach for moving beyond classic phylogeographic research. We use sequence capture probes and Illumina sequencing to generate data from >400 loci in order to infer the phylogeographic history of Salix melanopsis, a riparian willow with a disjunct distribution in coastal and the inland Pacific Northwest. We evaluate a priori phylogeographic hypotheses using coalescent models for parameter estimation, and the results support earlier findings that identified post-Pleistocene dispersal as the cause of the disjunction in S. melanopsis. We also conduct a series of model selection exercises using IMa2, Migrate-n and ∂a∂i. The resulting ranking of models indicates that refugial dynamics were complex, with multiple regions in the inland regions serving as the source for postglacial colonization. Our results demonstrate that new sources of data and new approaches to data analysis can rejuvenate phylogeographic research by allowing for the identification of complex models that enable researchers to both identify and estimate the most relevant parameters for a given system.