Biotinylated DNA Probes Research Articles

An Electrochemical Bioplatform Based on Acrylamide‐Induced Inhibition of DNA‐RNA Hybridization for the Determination of Acrylamide

AbstractThis study reports the development of an innovative electrochemical bioplatform for the indirect determination of acrylamide (AM) based on its inhibitory effect on the hybridization of nucleic acids through the formation of AM‐guanine single‐stranded (ssDNA) adducts. The method combines the advantages of the formation and selective immunorecognition of DNA/RNA heteroduplex, with the use of MBs and amperometric transduction using disposable SPCEs. Increasing concentrations of acrylamide prevented the efficient hybridization of a synthetic biotinylated DNA probe immobilized on streptavidin‐coated magnetic beads with its complementary RNA target and the as‐formed heteroduplex recognition with a specific antibody and a secondary HRP‐labeled antibody used for electrochemical readout. The resulting magnetic bioconjugates were magnetically trapped on the surface of the working electrode of a SPCE and the amperometric transduction was carried out in the presence of the H2O2/HQ system, recording a cathodic response inversely proportional to the AM concentration. The developed bioplatform is more competitive with other reported methods in terms of simplicity and assay time, allowing the selective detection of 1 nM AM in 60 min. In addition, the bioplatform was applied to the analysis of potato crisp water extracts.

Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms.

This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay.

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.

The Optimization of a Label-Free Electrochemical DNA Biosensor for Detection of Sus scrofa mtDNA as Food Adulterations

Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a label-free electrochemical DNA biosensor method was developed for the detection of pork in processed meat samples. Gold electrodeposited screen-printed carbon electrodes (SPCEs) were used and characterized using SEM and cyclic voltammetry. A biotinylated probe DNA sequence of the Cyt b S. scrofa gene mtDNA used as a sensing element containing guanine substituted by inosine bases. The detection of probe-target DNA hybridization on the streptavidin-modified gold SPCE surface was carried out by the peak guanine oxidation of the target using differential pulse voltammetry (DPV). The optimum experimental conditions of data processing using the Box–Behnken design were obtained after 90 min of streptavidin incubation time, at the DNA probe concentration of 1.0 µg/mL, and after 5 min of probe-target DNA hybridization. The detection limit was 0.135 µg/mL, with a linearity range of 0.5–1.5 µg/mL. The resulting current response indicated that this detection method was selective against 5% pork DNA in a mixture of meat samples. This electrochemical biosensor method can be developed into a portable point-of-care detection method for the presence of pork or food adulterations.

A Fluorescent Biosensor for Streptavidin Detection Based on Double-Hairpin DNA-Templated Copper Nanoparticles

In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.

Streamlined Purification of RNA-Protein Complexes Using UV Cross-Linking and RNA Antisense Purification.

RNA-protein interactions are important in development and disease, but identification of novel RNA-protein interactions remains challenging. Here, we describe an updated capture method to identify direct and specific RNA-protein interactions. First, RNA and protein are covalently cross-linked in living cells by treatment with UV light at 254 nanometers wavelength. The antisense purification approach is dependent upon nucleic acid hybridization between biotinylated DNA probes and a target RNA. Target protein:RNA:DNA complexes are enriched by capture on streptavidin magnetic beads and purified through several denaturing washes that remove nonspecific protein and nucleic acid interactors. Mass spectrometry is used to identify proteins that are specifically enriched in the target RNA capture. This method has been applied to discover the protein interactions of noncoding RNAs but can be used to capture any RNA where the target sequence is known.

Mild cognitive impairment is associated with low copy number of ribosomal genes in the genomes of elderly people.

Introduction: Mild cognitive impairments (MCI) accompanying aging are associated with oxidative stress. The ability of cells to respond to stress is determined by the protein synthesis level, which depends on the ribosomes number. Ribosomal deficit was documented in MCI. The number of ribosomes depends, together with other factors, on the number of ribosomal genes copies. We hypothesized that MCI is associated with low rDNA CN in the elderly person genome. Materials and Methods: rDNA CN and the telomere repeat (TR) content were determined in the DNA of peripheral blood leukocytes of 93 elderly people (61–91 years old) with MCI and 365 healthy volunteers (16–91 years old). The method of non-radioactive quantitative hybridization of DNA with biotinylated DNA probes was used for the analysis. Results: In the MCI group, rDNA CN (mean 329 ± 60; median 314 copies, n = 93) was significantly reduced (p < 10–15) compared to controls of the same age with preserved cognitive functions (mean 412 ± 79; median 401 copies, n = 168) and younger (16–60 years) control group (mean 426 ± 109; median 416 copies, n = 197). MCI is also associated with a decrease in TR DNA content. There is no correlation between the content of rDNA and TR in DNA, however, in the group of DNA samples with rDNA CN > 540, TR content range was significantly narrowed compared to the rest of the sample. Conclusion: Mild cognitive impairment is associated with low ribosomal genes copies in the elderly people genomes. A low level of rDNA CN may be one of the causes of ribosomal deficit that was documented in MCI. The potential possibilities of using the rDNA CN indicator as a prognostic marker characterizing human life expectancy are discussed.

Fully automated microRNA quantification technique based on bioluminescent enzyme immunoassay

MicroRNAs (miRNAs) are potential clinical biomarkers for the detection of various diseases. However, their quantification has not been implemented in clinical practice given the inconsistencies in their variable recovery rate and accuracy of the results. Thus, we utilized a technique based on bioluminescent enzyme immunoassay (BLEIA) to perform fully automated miRNA quantification using magnetic particles conjugated with antibodies targeting DNA-RNA hybrids, biotinylated DNA probes specific to miRNAs, and firefly luciferase-labeled streptavidin. This method enabled direct use of diluted serum and automation of all processes within 1 hr. The results revealed a wide linear range between 10 fmol/L and 1 nmol/L, high sensitivity with a detection limit of 6.3 fmol/L or below, high specificity with a false positive rate under 2.4%, and high reproducibility in intra- and inter-experimental observations (under CV 10% and r = 0.9965, respectively). Furthermore, a significant correlation was revealed between quantitative reverse transcription-polymerase chain reaction and BLEIA assay for the quantification of synthetic miRNA (r = 0.9993) and endogenous miRNA in healthy serums (r = 0.8203), respectively. Overall, we developed a fully automated miRNA quantification method based on BLEIA, which can be adopted in a clinical setting. However, further studies are needed to assess its clinical performance.

Engineered tracrRNA for enabling versatile CRISPR-dCas9-based biosensing concepts

In recent years, CRISPR-Cas (stands for: clustered regularly interspaced short palindromic repeats - CRISPR associated protein) based technologies have gained increasing attention in the biosensing field. Thanks to excellent sequence specificity, their use is of particular interest for detecting nucleic acid (NA) targets. In this context, signal generation and amplification can be realized by employing the cis-cleavage activity of the Cas9 protein, although other options involving the catalytically inactive dead Cas9 (dCas9) are increasingly explored. The latter are however mostly based on complex protein engineering processes and often lack efficient signal amplification. Here we showed for the first time that flexible signal generation and amplification properties can be integrated into the CRISPR-dCas9 complex based on a straightforward incorporation of a DNA sequence into the trans-activating CRISPR RNA (tracrRNA). The intrinsic nuclease activity of the engineered complex remained conserved, while the incorporated DNA stretch enabled two modes of amplified fluorescent signal generation: (1) as an RNA-cleaving DNA-based enzyme (DNAzyme) or (2) as hybridization site for biotinylated DNA probes, allowing subsequent enzyme labeling. Both signal generation strategies were demonstrated in solution as well as while coupled to a solid surface. Finally, in a proof of concept bioassay, we demonstrated the successful detection of single stranded DNA on magnetic microbeads using the engineered CRISPR-dCas9 complex. Thanks to the flexibility of incorporating different NA-based signal generation and amplification strategies, this novel NA engineering approach holds enormous promise for many new CRISPR-based biosensing applications.

A multipurpose biocapture nanoplatform based on multiwalled-carbon nanotubes non-covalently functionalized with avidin: Analytical applications for the non-amplified and label-free impedimetric quantification of BRCA1

We introduce an innovative and versatile multipurpose biocapture nanoplatform made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with avidin (Av) as support to immobilize a biorecognition element just by mixing them in solution. As proof-of-concept of the advantages of this platform, we propose a non-amplified and label-free impedimetric BRCA1 biosensor prepared by immobilization of a biotinylated DNA probe (bDNAp) at MWCNTs-Av nanoplatform and further deposition of the resulting biosensing layer at glassy carbon (GCE) or screen printed carbon (SPCE) electrodes. The hybridization event was detected from the changes in the charge transfer resistance (Rct) of [Fe(CN)6]3-/4-. The resulting genosensor made possible the fast, highly sensitive and selective quantification of BRCA1 in a simple way, with a linear range between 1.0 fM and 10 nM, a sensitivity of (460 ± 9) ΩM−1 (r2 = 0.997), a detection limit of 330 aM and a quantification limit of 1.0 fM. The reproducibility was 5.2% and 4.6% for 1.0 × 10−12 M and 1.0 × 10−14 M BRCA1, respectively, using the same MWCNTs-Av-bDNAp platform, and 5.2% for 1.0 × 10−12 M BRCA1 using 5 different platforms. The biosensor was challenged with fully non-complementary and mismatch DNA sequences, and mixtures of them, without interference. The detection of BRCA1 in enriched diluted human serum samples showed excellent recovery percentages.

Surface plasmon resonance biosensor for the detection of miRNAs by combining the advantages of homogeneous reaction and heterogeneous detection

The hybridization and enzymolysis reactions for nucleic acid detection were carried out on the chip surface in the traditional surface plasmon resonance (SPR) biosensors. Herein, we proposed an innovative method for microRNA (miRNA) detection in which the hybridization-enzymolysis recycling reactions were performed in solution. Duplex-specific nuclease (DSN) and streptavidin-modified gold nanoparticles (SA-AuNPs) were employed for enhancing the assay sensitivity. In the absence of miRNA, the biotinylated DNA probe (bio-DNA-bio, biotin tags at both the 3′ and 5′ termini of DNA) was attached to the SA-modified chip through the SA-biotin binding, allowing the capture of SA-AuNPs with the same interaction. As a result, a larger SPR signal was attained. However, in the presence of miRNA, bio-DNA-bio hybridized with miRNA was digested by DSN. In this process, the miRNA strand remained intact and participated in the next hybridization-enzymolysis recycling process. Thus, one miRNA could promote the hydrolysis of many bio-DNA-bio probes and allow the generation of numerous bio-DNA fragments. Meanwhile, the produced bio-DNA competed with the undigested bio-DNA-bio to bind SA on the chip surface. The digestion of bio-DNA-bio and the competitive binding between bio-DNA-bio and bio-DNA led to the attachment of fewer SA-AuNPs and then smaller SPR signals. The change in SPR signal at the concentration as low as 1 fM miRNA has been readily determined. The strategy possessed the advantageous properties of simple operation, fast response, high sensitivity and excellent specificity, serving as a viable means for the fabrication of novel sensing platforms.

Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners.

RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), controlling key cellular processes, including gene expression and translation of proteins. Current methodologies aimed at identifying and characterizing protein binding partners of specific RNAs of interest typically rely on tagging of the RNA with affinity aptamers, using in vitro transcribed RNA or immobilized oligonucleotides to capture RNA-protein complexes under native conditions. These assays are coupled with mass spectrometry or Western Blot analysis to identify or/and confirm interacting proteins. Here, we describe an alternative approach to identify protein binding partners of mRNAs and large long noncoding RNAs. This approach relies on biochemical pulldown of specific target RNAs and interacting protein partners from cellular lysates coupled with mass spectrometry to identify novel interacting proteins. By using 24-48 ~20 mer biotinylated DNA probes that hybridize to the target RNA, the method ensures high specificity and minimal off target binding. This approach is reproducible and fast and serves as a base for discovery studies to identify proteins that bind to RNAs of interest.

HIV-1 DNA-capture-seq is a useful tool for the comprehensive characterization of HIV-1 provirus

Regardless of recent advances in the development of anti-retroviral drugs, it is still extremely difficult to eradicate HIV-1 from infected individuals. The characterization of the HIV-1 provirus, a type of viral reservoir, with a high resolution is key to HIV-1 cure research. Here, we demonstrate that DNA-capture-seq is a powerful tool to obtain comprehensive information on the HIV-1 provirus. We use biotinylated DNA probes targeting the entire HIV-1 sequence to capture fragments containing HIV-1 sequences from DNA-seq libraries prepared for high throughput sequencing. We demonstrate that the protocol provided the entire proviral sequence from the beginning of the 5′ LTR to the end of the 3′ LTR. Since HIV-1 DNA-probes can hybridize not only viral fragments but also virus-host chimeric ones, the viral integration site information can also be obtained. We verify the efficiency of the protocol by using latently infected cell lines, such as ACH-2 and J1.1, and newly generated ones. The results reveal that the 2 new clones that we analyse harbour one copy of replication-competent provirus, suggesting that latency is not caused by genetic mutations or deletions of the provirus. In conclusion, HIV-1 DNA-capture-seq is a powerful tool to characterize the HIV-1 provirus at a single nucleotide resolution and therefore might be useful for various experiments aiming for an HIV-1 cure.

Recombinant Fusion Streptavidin as a Scaffold for DNA Nanotetrads for Nucleic Acid Delivery and Telomerase Activity Imaging in Living Cells.

Efficient platforms for intracellular delivery of nucleic acids are essential for biomedical imaging and gene regulation. We develop a recombinant fusion streptavidin as a novel protein scaffold for DNA nanotetrads for highly efficient nucleic acid delivery and telomerase activity imaging in living cells via cross-linking hybridization chain reaction (cHCR). The recombinant streptavidin protein is designed to fuse with multiple SV40 NLS (nuclear localization signal) and NES (nuclear export signal) domains and prepared through Escherichia coli expression. The recombinant NLS-SA protein allows facile assembly with four biotinylated DNA probes via high-affinity noncovalent interactions, forming a well-defined DNA tetrad nanostructure. The DNA nanotetrads are demonstrated to confer efficient cytosolic delivery of nucleic acid via a caveolar mediated endocytosis pathway, allowing efficient escape from lysosomal degradation. Moreover, the nanotetrads enable efficient cHCR assembly in response to telomerase in vitro and in cellulo, affording ultrasensitive detection and spatially resolved imaging for telomerase with a detection limit as low as 90 HeLa cells/mL. The fluorescence brightness obtained in live cell imaging is found to be dynamically correlated to telomerase activity and the inhibitor concentrations. Therefore, the proposed strategy may provide a highly efficient platform for nucleic acid delivery and imaging of biomarkers in living cells.

Colorimetric Detection of DNA Strands on Cellulose Microparticles Using ZZ-CBM Fusions and Gold Nanoparticles.

Nucleic acid testing requires skilled personnel and expensive instrumentation. A method for the colorimetric detection of oligonucleotides that combines cellulose microparticles with biomolecular recognition is presented. DNA sequences from Trypanosoma brucei and dengue are used as model targets. Cellulose microparticles (≈20 µm) are bioactived by anchoring anti-biotin antibodies via fusions that combine a carbohydrate-binding module (CBM) with the ZZ fragment of protein A. Samples are prepared by incubating DNA probes immobilized on ≈14 nm gold nanoparticles (AuNPs) with biotin-labeled targets and mixed with bioactive microparticles. The presence of unlabeled targets could also be probed by introducing a second, biotinylated DNA probe. The target:probe-AuNP hybrids are mixed with and captured by the microparticles, which change color from white to red. Depletion of AuNPs from the liquid is also signaled by a decrease in absorbance at 525 nm. It was possible to detect targets with concentrations as low as 50 n m. In the presence of noncomplementary targets, microparticles remain white and the liquid remains red. The system is able to discriminate targets with a high degree of homology (≈53%). Overall, it is demonstrated that simple systems for the visual detection of nucleic acids can be set up by combining cellulose microparticles with biomolecular recognition agents based on CBMs and AuNPs.

High-sensitive immunosensing of protein biomarker based on interfacial recognition-induced homogeneous exponential transcription

A novel and versatile immunosensing strategy was developed for ultrasensitive and specific detection of proteins by organically integrating interfacial specific target recognition and homogeneous transcription amplification. In principle, classic antigen-antibody sandwich structure on the microplate could realize the specific identification of target protein. Biotinylated DNA probe was subsequently introduced by streptavidin-biotin system as a bridge linking interfacial and homogeneous reaction. The biotinylated DNA initiated exponential transcription amplification in the solution, which converted per target recognition event on the interface to numerous single-stranded RNA products in solution for highly sensitive fluorescence immunosensing. The proposed immunoassay based on interfacial recognition-induced homogeneous exponential transcription (IR-HET) for vascular endothelial growth factor (VEGF) detection showed a good linear range from 0.01 to 1000 pg/mL and the limit of detection as low as 1 fg/mL, which was 3 orders lower than traditional ELISA method. The established strategy was also successfully applied to directly detect VEGF from culture supernatants of tumor cells and clinical body fluid samples, proving very high sensitivity, selectivity and low matrix effect. Therefore, IR-HET-based immunosensing strategy might become a potential powerful tool be applied in ultrasensitive detection of low abundance protein biomarker for clinical early diagnosis, treatment and prognosis.

Role of Somatic Mutations and Clonal Thrombopoiesis in Immune Thrombocytopenia

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disorder caused by antibodies directed at self-antigens leading to platelet destruction. Autoantibodies from most patients with ITP are directed to platelet membrane glycoprotein(GP) IIb-IIIa or GPIb-IX. 80% of ITP cases are classified as primary and it's unclear why auto reactive antibodies are formed in these patients. Studies from the United States, Asia and Europe show an increasing incidence of ITP with aging. Thrombopoietin receptor agonists (TPO-RA) interact directly with the TPO receptor on megakaryocytes to stimulate platelet production and can surprisingly produce sustained remissions of chronic ITP in some patients even after discontinuation of treatment. The heterogeneity of the course of ITP and the response to treatment, especially the response to treatment with thrombopoietin receptor agonists (TPO-RA), suggests pathogenic and clinical diversity of ITP. Clonal hematopoiesis is known to occur in elderly patients and has been associated with adverse outcomes including hematologic cancers and cardiovascular disease. We hypothesized that clonal hematopoiesis and associated passenger somatic mutations contribute to loss of immune tolerance in some ITP patients, causing the generation of auto-reactive antibodies and affecting the response to TPO-RA.METHODS: Platelet RNA was extracted from 13 blood samples obtained from 11 ITP patients using Direct-zolTM RNA MicroPrep Kit (Zymo Research). 2 patients provided samples during both relapse and remission. cDNA generation was performed from enriched total RNA using SMARTerR Target RNA Capture for IlluminaR Kit (Clontech). Target-specific biotinylated DNA probes were designed to hybridize to mRNA transcripts for common platelet antigens including (GP) IIb-IIIa and GPIb-IX. cDNA libraries were prepared using the Nextera™ XT DNA Library preparation kit. Following the pooling of libraries they were sequenced on an Illumina Nextseq 500 instrument in Mid-output mode with single-ended reads of 150 bp.RESULTS: In one of the paired patient specimens, we found substantial differences in platelet populations between relapse and remission on a TPO-RA. Different previously-reported non-disease-causing heterozygous single nucleotide variants (SNVs) were detected during relapse and remission. Expression at a level approximating 50% in remission and a reciprocal expression in relapse suggests that 2 point mutations leading to a missense amino acid change (Glu to Lys at amino acid 671) in GPIIb (ITGA2B) and (Thr to Met at amino acid 161) in GPIb (GP1BA) provoke an immune response (Table 1). Further, the reciprocal expression at these levels is consistent with expression of a somatic mutation in a clonal fashion.CONCLUSION: We documented that unique platelet clones were expressed during relapse and remission of ITP. These findings demonstrate that clonal expression of platelets bearing somatic mutations in target glycoproteins are associated with ITP disease course and response to therapy. Such somatic mutations are likely to be passenger mutations acquired prior to clonal expansion. Expression of a somatic mutation in a clonal fashion may drive autoimmune response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Colorimetric human papillomavirus DNA assay based on the retardation of avidin-induced aggregation of gold nanoparticles.

A colorimetric assay for human papillomavirus (HPV) DNA was developed based on the retardation of the avidin-induced aggregation of gold nanoparticles (AuNPs) by HPV DNA. Positively charged avidin acts as a coagulant for AuNP aggregation. In the presence of the target DNA, however, the aggregation of AuNPs is retarded owing to electrosteric stabilization as a result of the hybridization of the target and probe DNA. In the absence of HPV DNA, the stabilization effect caused by the biotinylated probe DNA is weak, resulting in NP aggregation and acolor change from red to purple. Aggregation may be easily observed with bare eyes or spectrophotometrically at about 560nm. The visual detection limit is 1nM. The assay was used for the determination of HPV DNA after polymerase chain reaction (PCR) amplification without any further purification. Graphical abstract Schematic presentation of the avidin-induced aggregation of unmodified gold nanoparticles (AuNPs) which leads to a color change from red to purple. In the presence of dsDNA, however, the aggregation is remarkably retarded.

Near-infrared fluorescent northern blot.

Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.

Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA)

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture.