Oligonucleotide Probes Research Articles

Karyotype establishment and development of specific molecular markers of Aegilops geniculata Roth based on SLAF-seq

The constant evolution of pathogens poses a threat to wheat resistance against diseases, endangering food security. Developing resistant wheat varieties is the most practical approach to circumventing this problem. As a close relative of wheat, Aegilops geniculata, particularly accession SY159 has evolved numerous beneficial traits that could be applied to improve wheat. In this study, we established the karyotype of SY159 by fluorescence in situ hybridization (FISH) using oligonucleotide probes Oligo-pTa535 and Oligo-pSc119.2 and a complete set of wheat-Ae. geniculata accession TA2899 addition lines as a reference. Using specific-locus amplified fragment sequencing (SLAF-seq) technology, 400 specific markers were established for detecting the SY159 chromosomes with efficiencies reaching 81.5%. The SY159-specific markers were used for classification of different homologous groups of SY159 against wheat-Ae. geniculata addition lines. We used these specific markers on the 7Mg chromosome after classification, and we successfully confirmed their suitability for studying different chromosomes of SY159. This study provides a foundation for accelerating the application of SY159 for genetic breeding programs designed to improve wheat.

Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems.

Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection.

Nanoribbon Biosensor-Based Detection of microRNA Markers of Prostate Cancer.

Prostate cancer (PC) is one of the major causes of death among elderly men. PC is often diagnosed later in progression due to asymptomatic early stages. Early detection of PC is thus crucial for effective PC treatment. The aim of this study is the simultaneous highly sensitive detection of a palette of PC-associated microRNAs (miRNAs) in human plasma samples. With this aim, a nanoribbon biosensor system based on "silicon-on-insulator" structures (SOI-NR biosensor) has been employed. In order to provide biospecific detection of the target miRNAs, the surface of individual nanoribbons has been sensitized with DNA oligonucleotide probes (oDNA probes) complementary to the target miRNAs. The lowest concentration of nucleic acids, detectable with our biosensor, has been found to be 1.1 × 10-17 M. The successful detection of target miRNAs, isolated from real plasma samples of PC patients, has also been demonstrated. We believe that the development of highly sensitive nanotechnology-based biosensors for the detection of PC markers is a step towards personalized medicine.

Precise Identification and Profiling of Surface Proteins of Ultra Rare Tumor Specific Extracellular Vesicle with Dynamic Quantitative Plasmonic Imaging.

Specific detection of tumor-derived EVs (tEVs) in plasma is complicated by nontumor EVs and non-EV particles. To accurately identify tEVs and profile their surface protein expression at single tEV resolution directly with clinical plasma is still an unmet need. Here, we present a Dynamic Immunoassay for Single tEV surface protein Profiling (DISEP), a kinetic assay based on surface plasmon resonance microscopy (SPRM) for specific single tEV profiling. DISEP adopts a pair of low-affinity oligonucleotide probes to respectively label EV surface proteins and functionalize an SPRM biosensor interface. tEVs labeled with the oligonucleotide probes possess distinctive binding kinetics from nonspecific particles in plasma, which permits accurate digital plasmonic counting of single EVs. We demonstrate DISEP for recognizing target EVs among 350-fold background plasma particles with high sensitivity (4677 EVs per μL). Clinical plasma samples were analyzed to discriminate between pancreatic cancer patients (n = 40) and healthy donors (n = 45). With a panel of biomarker signatures (EpCAM, HER2, and GPC1), DISEP only requires 10 μL primary sample from each donor to classify tumor patients with an area under the curve of 0.98. DISEP provides a highly specific EV detection and surface protein profiling strategy for early cancer diagnosis.

Electrochemical miRNA-34a-based biosensor for the diagnosis of Alzheimer’s disease

Alzheimer's disease (AD) is the most common dementia type and a leading cause of death and disability in the elderly. Diagnosis is expensive and invasive, urging the development of new, affordable, and less invasive diagnostic tools. The identification of changes in the expression of non-coding RNAs prompts the development of diagnostic tools to detect disease-specific blood biomarkers. Building on this idea, this work reports a novel electrochemical microRNA (miRNA) biosensor for the diagnosis of AD, based on carbon screen-printed electrodes (C-SPEs) modified with two gold nanostructures and a complementary anti-miR-34a oligonucleotide probe. This biosensor showed good target affinity, reflected on a 100 pM to 1 μM linearity range and a limit of detection (LOD) of 39 pM in buffer and 94 aM in serum. Moreover, the biosensor’s response was not affected by serum compounds, indicating selectivity for miR-34a. The biosensor also detected miR-34a in the cell culture medium of a common AD model, stimulated with a neurotoxin to increase miR-34a secretion. Overall, the proposed biosensor makes a solid case for the introduction of a novel, inexpensive, and minimally invasive tool for the early diagnosis of AD, based on the detection of a circulating miRNA overexpressed in this pathology.

Association of HLA-DRB1 alleles with anti-D alloimmunization in RhD negative pregnant women in India

BackgroundHuman leukocyte antigen (HLA) restriction plays an important role in the susceptibility to alloimmunization against red blood cell (RBC) antigens. The prevalence of anti-D alloimmunization in RhD negative pregnancy is still quite high in our population. Thus, we planned this study to determine the association of HLA-DRB1 alleles with anti-D alloimmunization in RhD negative pregnant women. Material and methodsRBC antibody screen (ABS) was performed for RhD negative pregnant women attending the antenatal clinic our institute. Those with a negative result were included in the ‘non-alloimmunized’ (NAL) group (‘Control’ group), while those with anti-D alloantibody on performing antibody identification were included in the alloimmunized (AL) group of the study (n = 50 each). ABS and identification were done using column agglutination technique. The HLA-DRB1 typing was done by Luminex based reverse sequence specific oligonucleotide probing (SSOP) using commercial kits. The HLA-DRB1 allele frequency was compared in both the groups. ResultsThere was a significant difference between the two groups in terms of gravida (p < 0.001) and history of anti-D immunoprophylaxis (p < 0.001). The frequency of HLA-DRB1*03 and HLA-DRB1*04 alleles was significantly higher in the AL group than the NAL group: 40 % versus 18 % [Odds Ratio (OR): 3.04, 95 % CI: 1.21–7.6; p = 0.015] for HLA-DRB1*03 alleles and 18 % versus 4 % (OR: 5.27, 95 % CI: 1.08–25.78, p = 0.025) for HLA-DRB1*04 alleles. ConclusionThe frequency of HLADRB1*03 and HLADRB1*04 alleles was significantly higher in RhD negative pregnant women alloimmunized with anti-D alloantibody.

Functionalized nanopores based on hybridization chain reaction: Fabrication and microRNA sensing

Enzyme-free hybridization chain reaction (HCR) technology is often used as a signal amplification tool for the detection of different targets. In this study, an ultrasensitive and label-free method for detecting miRNA-21 was developed using the nanopore ionic current rectification (ICR) technology coupled with HCR technology. The probe oligonucleotide (DNA1) was combined with the gold-coated nanopore through the Au–S bond to form a DNA1-functionalized gold-coated nanopore (DNA1-Au-coated nanopore). Since miRNA-21 is partially complementary to DNA1, it can be selectively recognized by DNA1-functionalized gold-coated nanopores. The target (miRNA-21) can induce the opening of hairpin DNA and HCR reaction after the introduction of hairpin DNA H1 and H2. The concentration of miRNA-21 will affect the combination of H1 and H2 on the inner wall of the nanopore, and its surface charge will change with the internal modification, thereby changing the ion current rectification ratio. Under the condition that the concentration of H1, H2 and HCR reaction time are constant, the change of ICR ratio is linearly correlated with the logarithm of miRNA-21 concentration within a certain range, which shows that the sensing strategy we designed can achieve target miRNA-21 detection. This ultrasensitive miRNA holds great promise in the field of cancer diagnosis.

The different subtelomeric structure among 1RS arms in wheat-rye 1BL.1RS translocations affecting their meiotic recombination and inducing their structural variation

BackgroundThe 1RS arm of wheat-rye 1BL.1RS translocations contains several subtelomeric tandem repeat families. To study the effect of the difference in the composition of these tandem repeats on the meiotic recombination of 1RS arms can help to enrich the genetic diversity of 1BL.1RS translocation chromosomes.ResultsFive wheat-rye 1BL.1RS translocation cultivars/lines were used to build two cross combinations including group 1 (20T401 × Zhou 8425B, 20T401 × Lovrin 10 and 20T401 × Chuannong 17) and group 2 (20T360-2 × Zhou 8425B, 20T360-2 × Lovrin 10 and 20T360-2 × Chuannong 17). Oligonucleotide (oligo) probes Oligo-s120.3, Oligo-TR72, and Oligo-119.2-2 produced the same signal pattern on the 1RS arms in lines 20T401 and 20T360-2, and another signal pattern in the three cultivars Zhou 8425B, Lovrin 10 and Chuannong 17. The Oligo-pSc200 signal disappeared from the 1RS arms of the line 20T401, and the signal intensity of this probe on the 1RS arms of the line 20T360-2 was weaker than that of the three cultivars. The five cultivars/lines had the same signal pattern of the probe Oligo-pSc250. The recombination rate of 1RS arms in group 1 was significantly lower than that in group 2. In the progenies from group 1, unequal meiotic recombination in the subtelomeric pSc119.2 and pSc250 tandem repeat regions, and a 1BL.1RS with inversion of 1RS segment between the pSc200 and the nucleolar organizer region were found.ConclusionsThis study provides a visual tool to detect the meiotic recombination of 1RS arms. The meiotic recombination rate of 1RS arms was affected by the variation of pSc200 tandem repeat, indicating the similar composition of subtelomeric tandem repeats on these arms could increase their recombination rate. These results indicate that the 1RS subtelomeric structure will affect its recombination, and thus the localization of genes on 1RS by means of meiotic recombination might also be affected.

The 18S rRNA genes of Haemoproteus (Haemosporida, Apicomplexa) parasites from European songbirds with remarks on improved parasite diagnostics

BackgroundThe nuclear ribosomal RNA genes of Plasmodium parasites are assumed to evolve according to a birth-and-death model with new variants originating by duplication and others becoming deleted. For some Plasmodium species, it has been shown that distinct variants of the 18S rRNA genes are expressed differentially in vertebrate hosts and mosquito vectors. The central aim was to evaluate whether avian haemosporidian parasites of the genus Haemoproteus also have substantially distinct 18S variants, focusing on lineages belonging to the Haemoproteus majoris and Haemoproteus belopolskyi species groups.MethodsThe almost complete 18S rRNA genes of 19 Haemoproteus lineages of the subgenus Parahaemoproteus, which are common in passeriform birds from the Palaearctic, were sequenced. The PCR products of 20 blood and tissue samples containing 19 parasite lineages were subjected to molecular cloning, and ten clones in mean were sequenced each. The sequence features were analysed and phylogenetic trees were calculated, including sequence data published previously from eight additional Parahaemoproteus lineages. The geographic and host distribution of all 27 lineages was visualised as CytB haplotype networks and pie charts. Based on the 18S sequence data, species-specific oligonucleotide probes were designed to target the parasites in host tissue by in situ hybridization assays.ResultsMost Haemoproteus lineages had two or more variants of the 18S gene like many Plasmodium species, but the maximum distances between variants were generally lower. Moreover, unlike in most mammalian and avian Plasmodium species, the 18S sequences of all but one parasite lineage clustered into reciprocally monophyletic clades. Considerably distinct 18S clusters were only found in Haemoproteus tartakovskyi hSISKIN1 and Haemoproteus sp. hROFI1. The presence of chimeric 18S variants in some Haemoproteus lineages indicates that their ribosomal units rather evolve in a semi-concerted fashion than according to a strict model of birth-and-death evolution.ConclusionsParasites of the subgenus Parahaemoproteus contain distinct 18S variants, but the intraspecific variability is lower than in most mammalian and avian Plasmodium species. The new 18S data provides a basis for more thorough investigations on the development of Haemoproteus parasites in host tissue using in situ hybridization techniques targeting specific parasite lineages.

Assessment of genetic differences in animals as exemplified by representatives of the genus &lt;i&gt;Camelus&lt;/i&gt;

Data on the genetic variability of genomic DNA from two species of camels (Dromedary and Bactrian) are presented. It is noted that these animal species are of great importance in a number of southern countries, they are used as farm animals (milk, meat, wool), as draft, riding and sports animals. At present, much attention is paid to the study of camels in order to identify their genetic characteristics that can be used in breeding work. One of the research methods is multilocus analysis using labeled oligonucleotide probes. The latter selectively hybridize in separate regions of genomic DNA, leading to the appearance of specific genetic profiles characteristic of each individual. Labeling the probe with digoxigenin makes it possible to detect the results of hybridization on the filter. After the reaction of molecular hybridization of the probe with genomic DNA of camels, from 3 to 15 DNA fragments were detected, while the pattern of hybridization was very different in Dromedaries and Bactrians, which indicates a significant genetic difference in the organization of genomes. The coefficient of similarity of individuals in Bactrians was significantly higher than in Dromedaries (0.48 versus 0.39); interspecific similarity coefficient in this parameter was only 0.13. The calculation of the genetic distance between populations gave a rather high value of 0.305, which is significantly higher than the previously obtained data on cattle (from 0.05 to 0.10). Intrapopulation genetic diversity was assessed by the criterion of average heterozygosity. Calculations showed greater genetic diversity in the dromedary population (H = 0.72), which was indirectly confirmed by a lower similarity coefficient in this group of animals.

Therapeutic-oligonucleotides activated by nucleases (TOUCAN): A nanocarrier system for the specific delivery of clinical nucleoside analogues

Nucleoside analogues have been in clinical use since 1960s and they are still used as the first therapeutic option for several cancers and viral infections, due to their high therapeutic efficacy. However, their wide clinical acceptance has been limited due to their high toxicity and severe side effects to patients. Herein, we report on a nanocarrier system that delivers nucleosides analogues in a target-specific manner, making nucleoside-based therapeutics safer and with the possibility to be used in other human conditions. This system, named, Therapeutic OligonUCleotides Activated by Nucleases” (TOUCAN) combines: i) the recognition power of oligonucleotides as substrates, ii) the use of nucleases as enzymatic biomarkers and iii) the clinical efficacy of nucleoside analogues, in a single approach. As a proof-of-concept, we report on a TOUCAN that is activated by a specific nuclease produced by bacteria and releases a therapeutic nucleoside, floxuridine. We demonstrate, for the first time, that, by incorporating a therapeutic nucleoside analogue into oligonucleotide probes, we can specifically inhibit bacterial growth in cultures. In this study, Staphylococcus aureus was selected as the targeted bacteria and the TOUCAN strategy successfully inhibited its growth with minimal inhibitory concentration (MIC) values ranging from 0.62 to 40 mg/L across all tested strains. Moreover, our results indicate that the intravenous administration of TOUCANs at a dose of 20 mg/kg over a 24-h period is a highly effective method for treating bacterial infections in a mouse model of pyomyositis. Importantly, no signs of toxicity were observed in our in vitro and in vivo studies. This work can significantly impact the current management of bacterial infections, laying the grounds for the development of a different class of antibiotics. Furthermore, it can provide a safer delivery platform for clinical nucleoside therapeutics in any human conditions, such as cancer and viral infection, where specific nuclease activity has been reported.

Chemical Approaches To Investigate Post-transcriptional RNA Regulation.

RNA plays a central role in biological processes, and its activity is regulated by a host of diverse chemical and biochemical mechanisms including post-transcriptional modification and interactions with RNA-binding proteins. Here, we describe our efforts to illuminate RNA biology through the application of chemical tools, focusing on post-transcriptional regulatory mechanisms. We describe the development of an activity-based protein profiling approach for discovery and characterization of RNA-modifying enzymes. Next, we highlight novel approaches for RNA imaging based upon metabolic labeling with modified nucleosides and engineering of the nucleotide salvage pathway. Finally, we discuss profiling RNA-protein interactions using small molecule-dependent RNA editing and synthetic photo-cross-linkable oligonucleotide probes. Our work provides enabling technologies for deciphering the complexity of RNA and its diverse functions in biology.

Photoinduced electron transfer detection method for identifying UGT1A1*28 microsatellites.

During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at the end of a DNA strand decreased upon hybridization with guanine (G). This phenomenon is referred to as photoinduced electron transfer (PeT). Using this phenomenon, we devised a method for the naked-eye detection of UGT1A1*28 (thymine-adenine (TA)-repeat polymorphism). Fluorescently labeled single-stranded DNA (ssDNA) oligonucleotides (probes) were designed and hybridized with complementary strand DNAs (target DNAs). Base pair formation at the blunt end between fluorescently labeled C (probe side) and G (target side), induced dramatic fluorescence quenching. Additionally, when the labeled-CG pair formed near the TA-repeat sequence, different TA-repeat numbers were discriminated. However, obtaining enough target DNA for this probe by typical polymerase chain reaction (PCR) was difficult. To enable the practical use of the probe, producing sufficient target DNA remains problematic.

Oligonucleotide Fluorescence In Situ Hybridization: An Efficient Chromosome Painting Method in Plants.

Fluorescence in situ hybridization (FISH) is an indispensable technique for studying chromosomes in plants. However, traditional FISH methods, such as BAC, rDNA, tandem repeats, and distributed repetitive sequence probe-based FISH, have certain limitations, including difficulties in probe synthesis, low sensitivity, cross-hybridization, and limited resolution. In contrast, oligo-based FISH represents a more efficient method for chromosomal studies in plants. Oligo probes are computationally designed and synthesized for any plant species with a sequenced genome and are suitable for single and repetitive DNA sequences, entire chromosomes, or chromosomal segments. Furthermore, oligo probes used in the FISH experiment provide high specificity, resolution, and multiplexing. Moreover, oligo probes made from one species are applicable for studying other genetically and taxonomically related species whose genome has not been sequenced yet, facilitating molecular cytogenetic studies of non-model plants. However, there are some limitations of oligo probes that should be considered, such as requiring prior knowledge of the probe design process and FISH signal issues with shorter probes of background noises during oligo-FISH experiments. This review comprehensively discusses de novo oligo probe synthesis with more focus on single-copy DNA sequences, preparation, improvement, and factors that affect oligo-FISH efficiency. Furthermore, this review highlights recent applications of oligo-FISH in a wide range of plant chromosomal studies.

Designing of rapid assay for the detection of RdRp/Orf1ab specific to SARS-CoV-2

SARS-CoV-2 is still threat and mostly used detection method is real time reverse transcriptase polymerase chain reaction (rRT-PCR) for the open reading frame (Orf1ab), RNA-dependent RNA polymerase (RdRp), nucleocapsid (N) and envelope (E) genes of virus. However, rRT-PCR may have false negative rate for the nucleic acid detection. Since the RdRp/Orf1ab has high sensitivity for the molecular detection, two sandwich models, Model 1A-Model 1B, based on hybridization on lateral flow assay (LFA) were designed here and applied with the synthetic and clinical samples of RdRp/Orf1ab. In this purpose colloidal gold nanoparticles (AuNPs) were used as label. Membranes having different flow rate, three oligonucleotide probe concentrations and running buffers were used. Although synthetic target sequence was recognized by all the LFAs, PCR products obtained from either the synthetic plasmid DNA or oro/nasopharyngeal swabs were detected by Model 1 A using W12 membrane. Designed strip assays detected the RdRp/Orf1ab of the clinical samples as 100% sensitivity and specifity. It means that they might be used for the detection of virus and can be modified for the recognition of mutant genes of virus. These findings also demonstrated the importance of membranes, sandwich models, probe concentrations and sample contents for developing LFAs for viral detection.

Karyotype and Phylogenetic Relationship Analysis of Five Varieties and Cultivars of Zanthoxylum armatum Based on Oligo-FISH.

Green prickly ash (Zanthoxylum armatum) has edible and medicinal value and is an economically significant plant in many countries. Z. armatum has many cultivars and varieties with similar phenotypes that are difficult to distinguish via traditional methods. In this study, we utilized oligo-FISH to distinguish five varieties and cultivars of Z. armatum on the basis of three oligonucleotide probes of 5S rDNA, (AG3T3)3, and (GAA)6. Karyotype analysis of the five varieties and cultivars of Z. armatum showed that the Z. armatum 'Tengjiao' karyotype formula was 2n = 2x = 98m with karyotype type 1C and an arm ratio of 4.3237, including two pairs of 5S rDNA signals and five pairs of (GAA)6 signals. The karyotype formula of Z. armatum 'Youkangtengjiao' was 2n = 2x = 128m + 8sm with karyotype type 2B and an arm ratio of 3.5336, including three pairs of 5S rDNA signals and 17 pairs of (GAA)6 signals. The karyotype formula of Z. armatum var. novemfolius was 2n = 2x = 134m + 2sm with karyotype type 1C and an arm ratio of 5.5224, including two pairs of 5S rDNA signals and eight pairs of (GAA)6 signals. The karyotype formula of Z. armatum 'YT-03' was 2n = 2x = 2M + 128m + 4sm + 2st with karyotype type 2C and an arm ratio of 4.1829, including three pairs of 5S rDNA signals and nine pairs of (GAA)6 signals. The karyotype formula of Z. armatum 'YT-06' was 2n = 2x = 126m + 10sm with cytotype 2B and an arm ratio of 3.3011, including three pairs of 5S rDNA signals and two pairs of (GAA)6 signals. The five varieties and cultivars of Z. armatum had (AG3T3)3 signals on all chromosomes. The chromosomal symmetry of Z. armatum 'Tengjiao' was high, whereas the chromosomal symmetry of Z. armatum 'YT-03' was low, with the karyotypes of the five materials showing a trend toward polyploid evolution. The phylogenetic relationship between Z. armatum 'Tengjiao' and Z. armatum var. novemfolius was the closest, while that between Z. armatum 'YT-03' and Z. armatum 'YT-06' was closer than with Z. armatum 'Youkangtengjiao' according to oligo-FISH. The results provided a karyotype profile and a physical map that contributes to the distinction of varieties and cultivars of Z. armatum and provides strategies for distinguishing other cultivated species.

Proximity Ligation Assay (PLA) for Fillets of Drosophila Larvae.

The ability to detect protein-protein interactions is critical for understanding the mechanisms underlying protein and cell function. Current methods to assay protein-protein interactions, such as co-immunoprecipitation (Co-IP) and fluorescence resonance energy transfer (FRET), have limitations; for example, Co-IP is an in vitro technique and may not reflect the situation in vivo, and FRET typically suffers from low signal-to-noise ratio. The proximity ligation assay (PLA) is an in situ method for inferring protein-protein interaction with a high signal-to-noise ratio. The PLA technique can indicate that two different proteins are closely associated by the ability of two secondary antibody oligonucleotide probes to hybridize when they are close to each other. This interaction generates a signal with rolling-circle amplification using fluorescent nucleotides. Although a positive result does not indicate that two proteins directly interact, it implies a potential in vivo interaction that can then be verified in vitro. PLA uses primary antibodies against the two proteins (or epitopes) of interest, one raised in mouse and the other raised in rabbit. When these antibodies bind to proteins that lie within 40 nm of each other in the tissue, complementary oligonucleotides conjugated individually to mouse and rabbit secondary antibodies can anneal to form a template for rolling-circle amplification. Using fluorescently labeled nucleotides, rolling circle amplification generates a strong fluorescent signal in areas of the tissue where the two proteins are found together that is detected using conventional fluorescence microscopy. This protocol describes how to perform PLA in vivo on the central nervous system and peripheral nervous system of third-instar larvae of the fruit fly Drosophila melanogaster.

Colorimetric bacteria sensing of Pseudomonas aeruginosa using gold nanoparticle probes

BackgroundDue to the advantages of molecular methods over biochemical methods, the use of molecular methods for diagnosing nosocomial infections such as Pseudomonas can be an appropriate and rapid way to choose the right diagnosis and treatment of infection and prevent further complications caused by the infection. The present article provides a description of the development of a nanoparticle-based detection technique for sensitive and specific deoxyribonucleic acid-based diagnostic of Pseudomonas aeruginosa. Specific thiolated oligonucleotide probes for one of the hypervariable regions of the 16S rDNA gene were designed and applied for colorimetric detection of the bacteria. ResultsThe results of gold nanoprobe-nucleic sequence amplification indicated the probe attached to gold nanoparticles in the presence of the target deoxyribonucleic acid. It caused aggregation of gold nanoparticles in the form of connected networks resulting in color change and indicating the presence of the target molecule in the sample, which could be observed by the naked eye. In addition, the wavelength of gold nanoparticles changed from 524 to 558 nm. Multiplex polymerase chain reactions were performed using four specific genes of Pseudomonas aeruginosa (oprL, oprI, toxA, and 16S rDNA). The sensitivity and specificity of the two techniques were assessed. According to the observations, the specificity of both techniques was 100%, and the sensitivity was 0.5 ng/μL and 0.01 ng/μL of genomic deoxyribonucleic acid for multiplex polymerase chain reaction and colorimetric assay, respectively. ConclusionsThe sensitivity of colorimetric detection was about 50 times higher than the polymerase chain reaction using the 16SrDNA gene. The results of our study proved to be highly specific with potential use for early detection of Pseudomonas aeruginosa.

Role of probe design and bioassay configuration in surface enhanced Raman scattering based biosensors for miRNA detection

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100–10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays.

#5950 MAJOR HLA ALLELE AND HAPLOTYPE FREQUENCIES IN THE MALTESE KIDNEY TRANSPLANT REGISTRY

Abstract Background and Aims The human leukocyte antigens (HLA) are the most diverse and polymorphic genes in the human genome and matching them is of vital importance for maximising the survival of kidney transplants. The Maltese archipelago consists of three inhabited islands in the Mediterranean Sea, with a total population of just over half a million people. HLA polymorphisms in Malta have never been described. The aim of this study was to investigate allelic frequencies and determine the most common haplotypes for major HLA Class I and Class II groups in the Maltese kidney transplant registry. This will hopefully facilitate organ sharing with other European countries and improve the HLA matching for kidney transplant recipients. Method All patients of Maltese ethnicity on the national kidney transplant list between 2018 and 2022 were analysed retrospectively. HLA typing is routinely performed at medium to high resolution using polymerase chain reaction sequence specific oligonucleotide probes and Luminex technology at the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ and HLA-DP loci. Allele and haplotype frequencies were calculated by direct counting and simple percentages. Results A total of 191 patient samples were analysed yielding the following allele types: 16 HLA-A, 31 HLA-B, 17 HLA-C, 15 HLA-DRB1, 6 HLA-DP, 7 HLA-DQ, 5 HLA-DRB3, one HLA-DRB4 and one HLA DRB5. The most frequent allele groups for the HLA-A locus were HLA-A*02 (30.9%), HLA-A*24 (12.0%) and HLA-A*01 (11.8%) whilst for the HLA-B locus these were HLA-B*35 (16.2%), HLA-B*18 (11.5%) and HLA-B*51 (7.1%). The most prevalent allele groups for the HLA-DRB1 locus were HLA-DRB1*11 (24.1%), HLA-DRB1*04 (13.1%) and HLA-DRB1*07 (11.3%). The commonest allele group for each of HLA-C, HLA-DP and HLA-DR loci were HLA-C*07 (23.6%), HLA-DP4 (5.8%), HLA-DQ7 (31.2%) respectively. The five most frequently encountered haplotypes were HLA-A*02-B*13-DRB1*07 (2.1%), HLA-A*02-B*35-DRB1*04 (1.6%), HLA-A*02-B*18-DRB1*10 (1.3%), HLA-A*02-B*35-DRB1*11 (1.3%), HLA-A*24-B*35-DRB1*11 (1.3%). Conclusion This study provides the first data on the Major HLA allele and haplotype frequencies in Maltese kidney transplant candidates. Such data may be crucial in setting up international kidney exchange programmes.