Rapid detection of African swine fever virus via SERS probe-modified sandwich hybridization assay.

Rapid and reliable detection method for African swine fever virus (ASFV) is proposed by surface-enhanced Raman spectroscopy (SERS). The ASFV target DNA can be specifically captured by sandwich hybridization between nanomagnetic beads and a SERS probe. Experimental results show that the significant Raman signal of the SERS probe with gold nanoparticles and a molecular reporter DTNB (5,5'-dimercapto-bis (2-nitrobenzoic acid)) can be adopted for detecting the hybridization chain reaction of ASFV DNA. The advantage of the SERS sandwich hybridization assay is the large response range from the single molecule level to 108 copies per mL, which not only can overcome the tedious time required for the amplification reaction but also provides a comparative method to polymerase chain reaction. Furthermore, real samples of African swine fever virus were detected from different subjects of swine fever virus including porcine reproductive respiratory syndrome virus and Japanese encephalitis virus. The proposed biosensor method can rapidly detect ASFV correctly within 15 min as a simple, convenient, low-cost detection approach. The biosensor can be used as a platform for the determination in biological, food, and environmental analytical fields.

Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay.

BACKGROUND: Traditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control. METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/µl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China. RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/µl for S. aureus, 103 copies/µl for S. flexneri, 105 copies/µl for A. caviae, 105 copies/µl for V. vulnificus, 100 copies/µl for S. enterica, 105 copies/µl for P. vulgaris, and 100 copies/µl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species. CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.

Toward Continuous Molecular Testing Using Gold-Coated Threads as Multi-Target Electrochemical Biosensors.

Analytical systems based on isothermal nucleic acid amplification tests (NAATs) paired with electroanalytical detection enable cost-effective, sensitive, and specific digital pathogen detection for various in situ applications such as point-of-care medical diagnostics, food safety monitoring, and environmental surveillance. Self-assembled monolayers (SAMs) on gold surfaces are reliable platforms for electroanalytical DNA biosensors. However, the lack of automation and scalability often limits traditional chip-based systems. To address these challenges, we propose a continuous thread-based device that enables multiple electrochemical readings on a functionalized working electrode Au thread with a single connection point. We demonstrate the possibility of rolling the thread on a spool, which enables easy manipulation in a roll-to-roll architecture for high-throughput applications. As a proof of concept, we have demonstrated the detection of recombinase polymerase amplification (RPA) isothermally amplified DNA from the two toxic microalgae species Ostreopsis cf. ovata and Ostreopsis cf. siamensis by performing a sandwich hybridization assay (SHA) with electrochemical readout.

Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology.

Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.

A comparison between the FlowCam 8100, microscopy, and sandwich hybridization assay for quantifying abundances of the saxitoxin-producing dinoflagellate, Alexandrium catenella.

Light microscopy, FlowCam, and sandwich hybridization assay (SHA) are three approaches that facilitate the monitoring of harmful algal bloom (HAB) forming phytoplankton. Yet, cross-comparisons among these techniques have not been conducted. This study addressed that gap using the saxitoxin-producing 'red tide' dinoflagellate Alexandrium catenella, a species responsible for blooms and paralytic shellfish poisoning worldwide. To achieve this goal, the dynamic ranges of each technique were compared using A. catenella cultures spanning low (pre-bloom), moderate (bloom), and high (dense bloom) levels. To assess field detection, water samples containing very low (<3 cells mL-1) A. catenella levels were collected from Long Island Sound, USA (Jun-Aug 2021) and evaluated using each method. Field samples were also spiked with A. catenella to high (160 cells mL-1) or low (40 cells mL-1) concentrations. In general, microscopy, FlowCam, and SHA returned comparable A. catenella cell concentrations for all tests. Mean cell concentrations from laboratory intercalibration experiments were not significantly different for any method or concentration (ANOVA, p > 0.05). However, relative to microscopy at times SHA produced non-detect signals <2 cells mL-1 in field samples and the FlowCam slightly underestimated cell concentrations when A. catenella abundances were high in laboratory and field samples. Mean cell concentrations of spike experiments were not significantly different for any test date, sampling location, or method, despite variability among methods within the high concentration treatment (ANOVA, p > 0.05 for all treatments). Findings are relevant to HAB researchers, managers, and public health officials because they help reconcile disparate cell abundance datasets that inform numerical models and enhance HAB monitoring and prediction. Results are also likely broadly applicable to several HAB species.

Identification of single nucleotide polymorphisms by a peptide nucleic acid-based sandwich hybridization assay coupled with toehold-mediated strand displacement reactions.

In this work, we developed a simple and accurate peptide nucleic acid (PNA)-based sandwich hybridization assay for single nucleotide polymorphisms (SNPs) in the p53 gene. Our approach combines the enzyme-free toehold-mediated strand displacement reaction (SDR) with real-time enzyme-linked immunosorbent assay (ELISA) to detect SNPs with high sensitivity and specificity. A PNA-DNA heteroduplex with an external toehold is designed and fixed on well surface of a 96-well plate. The strand displacement from PNA-DNA heteroduplexes is initiated by the hybridization of target sequence with the toehold domain and ends with the fully displacing of the incumbent DNA. Finally, the as formed PNA-target DNA duplex with overhang at its 5'-end hybridizes with a biotin-labeled reporter PNA to form a sandwich structure on surface for signal amplification. The proposed PNA-based sandwich biosensor displays high sensitivity and greatly enhanced discriminability to target p53 gene segments against single-base mutant sequences compared to its all-DNA counterpart. Furthermore, the probe design is elegantly simple and the sensing procedure is easy to operate. We believe that this strategy may provide a simple and universal strategy for SNPs detection through easily altering the sequences of probes according to the sequences around target SNPs.

Sandwich Hybridization Assay for In Situ Real-Time Cyanobacterial Detection and Monitoring: A Review.

As cyanobacterial harmful algal bloom (cHAB) events increase in scale, severity, frequency, and duration around the world, rapid and accurate monitoring and characterization tools have become critically essential for regulatory and management decision-making. The composition of cHAB-forming cyanobacteria community can change significantly over time and space and be altered by sample preservation and transportation, making in situ monitoring necessary to obtain real-time and localized information. Sandwich hybridization assay (SHA) utilizes capture oligonucleotide probes for sensitive detection of target-specific nucleic acid sequences. As an amplification-free molecular biology technology, SHA can be adapted for in-situ, real-time or near real-time detection and qualitatively or semi-quantitatively monitoring of cHAB-forming cyanobacteria, owing to its characteristics such as being rapid, portable, inexpensive, and amenable to automation, high sensitivity, specificity and robustness, and multiplexing (i.e., detecting multiple targets simultaneously). Despite its successful application in the monitoring of marine and freshwater phytoplankton, there is still room for improvement. The ability to identify a cHAB community rapidly would decrease delays in cyanotoxin analyses, reduce costs, and increase sample throughput, allowing for timely actions to improve environmental and human health and the understanding of short- and long-term bloom dynamics. Real-time detection and quantitation of HAB-forming cyanobacteria is essential for improving environmental and public health and reducing associated costs. We review and propose to apply SHA for in situ cHABs monitoring.

Analyses of Bloodmeal Hosts and Prevalence of Rickettsia parkeri in the Gulf Coast Tick Amblyomma maculatum (Acari: Ixodidae) From a Reconstructed Piedmont Prairie Ecosystem, North Carolina.

Host feeding patterns and the prevalence of infection with Rickettsia parkeri were determined for the primary vector, Amblyomma maculatum Koch as well as sympatric tick species A. americanum (Linnaeus) and Dermacentor variabilis (Say) collected from a reconstructed prairie in the Piedmont region of North Carolina during 2011 and 2012. The occurrence of R. parkeri among A. maculatum adults and nymphs was 36.9% (45/122) and 33.3% (2/6), respectively. Rickettsia parkeri was detected in a single male A. americanum 2.3% (1/43). A PCR-reverse line blot hybridization assay of a 12S rDNA fragment amplified from remnant larval and nymphal bloodmeals of host-seeking ticks was used to identify bloodmeal hosts. Of the tick samples tested, bloodmeal host identification was successful for 29.3% (12/41) of adult A. americanum and 39.2% (20/51) of adult D. variabilis. For A. maculatum, bloodmeal host identification was successful for 50% (61/122) of adults collected from vegetation and 100% (4/4) of nymphs removed from cotton rats (Sigmodon hispidus Say and Ord). The cotton rat was the most common bloodmeal host with 59.0% (36/61) identified for adult A. maculatum. No statistically significant association was observed, however, between bloodmeal host and pathogen prevalence for any tick species. While the cotton rat was an important bloodmeal host for A. maculatum nymphs, this vertebrate did not appear to be the primary source of R. parkeri infection for A. maculatum.

Integrative analysis of key microRNA-mRNA complexes and pathways in aortic aneurysm.

Background: This study investigated the therapeutic targets of aortic aneurysm (AA) and provided insights into the pathogenesis and molecular mechanisms of AA. Methods: The messenger RNA (mRNA) datasets, GSE9106 (blood samples) and GSE7084 (tissue samples), and the microRNA (miRNA) datasets, GSE92427 (blood samples) and GSE110527 (tissue samples), were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DE-miRNAs) were analyzed by limma. Based on the co-DEGs and co-DE-miRNAs between the AA blood and tissue datasets, the miRNA-mRNA regulatory pairs were predicted. Functional enrichment analyses and gene set enrichment analysis (GSEA) were performed and the protein-protein interaction (PPI) network was generated to further analyze the related genes and their functions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and tyramide signal amplification (TSA)-in situ hybridization (ISH) assays were performed to detect the expression of co-DE-miRNAs in AA clinical tissue samples and normal aorta samples. Results: There were 19 upregulated and 5 downregulated co-differential mRNAs. MiR-4306 was the upregulated co-differential miRNA, and miR-3198 was the downregulated co-differential miRNA by blood-tissue co-analysis. Based on the co-DEGs and co-DE-miRNAs, 4 miRNA-mRNA regulatory pairs were predicted. PPI networks were constructed of co-DEGs with 6 relationship pairs. RT-qPCR and TSA-ISH assays showed the upregulation of miR-4306 and the downregulation of miR-3198 in AA tissue samples. Conclusions: This study provided evidence regarding the differential regulatory miRNA-mRNA networks in AA blood and tissue samples and identified key genes and signaling pathways related to AA, which provided insights into potential targets and mechanisms of AA pathogenesis and progression.

A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach.

Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10-100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.

Digital Hybridization Human Papillomavirus Assay with Attomolar Sensitivity without Amplification.

Detection of nucleic acid without amplification can avoid problems associated with thermal cycling such as labor-intensiveness and aerosol pollution. Here we develop a droplet-based digital microfluidic hybridization assay for nucleic acid detection with attomolar sensitivity. This assay provides a clinically useful sensitivity for detecting human papillomavirus (HPV) without amplification. The sensitivity is accomplished using femtoliter-sized droplet microfluidics for concentrating enzyme-catalyzed fluorescent products into a detectable signal and magnetic beads for accelerating reaction time. Meanwhile, using magnetic beads and droplet microfluidic chips, we can improve the sampling efficiency over conventional methods. We characterized the sensitivity, selectivity, detection range, stability, and accuracy of our assay. Our assay is 50-fold more sensitive than the traditional hybrid capture assay. The assay without amplification avoids problems of complex handling procedures and aerosol pollution. The direct and sensitive detection of nucleic acid using a droplet microfluidic system provides an early disease diagnosis tool.

Distribution and biotransformation of therapeutic antisense oligonucleotides and conjugates.

Drug properties of antisense oligonucleotides (ASOs) differ significantly from those of traditional small-molecule therapeutics. In this review, we focus on ASO disposition, mainly as characterized by distribution and biotransformation, of nonconjugated and conjugated ASOs. We introduce ASO chemistry to allow the following in-depth discussion on bioanalytical methods and determination of distribution and elimination kinetics at low concentrations over extended periods of time. The resulting quantitative data on the parent oligonucleotide, and the identification and quantification of formed metabolites define the disposition. Proper quantitative understanding of disposition is pivotal for nonclinical to clinical predictions, supports communication with health agencies, and increases the probability of delivering optimal ASO therapy to patients.

Development and comparison of cross-linking and non-crosslinking probe-gold nanoparticle hybridization assays for direct detection of unamplified bovine viral diarrhea virus-RNA.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a major economic disease that has been spread in most countries. In addition to vaccination, one of the main ways to control the disease and prevent it from spreading is to detect and cull infected animals, especially those with persistent infection (PI). We developed and compared two colorimetric biosensor assays based on probe-modified gold nanoparticles (AuNPs) to detect BVDV. Specific probes were designed to detect the 5' untranslated region of BVDV-RNA. The thiolated probes were immobilized on the surface of the AuNPs. Two methods of cross-linking (CL) and non-crosslinking (NCL) probe-AuNPs hybridization were developed and compared. RESULTS: The hybridization of positive targets with the two probe-AuNPs formed a polymeric network between the AuNPs which led to the aggregation of nanoparticles and color change from red to blue. Alternatively, in the NCL mode, the hybridization of complementary targets with the probe-AuNPs resulted in the increased electrostatic repulsion in nanoparticles and the increased stabilization against salt-induced aggregation. The CL and NCL assays had detection limits of 6.83 and 44.36 ng/reaction, respectively. CONCLUSION: The CL assay showed a higher sensitivity and specificity; in contrast, the NCL assay did not require optimizing and controlling of hybridization temperature and showed a higher response speed. However, both the developed methods are cost-effective and easy to perform and also could be implemented on-site or in local laboratories in low-resource countries.

PCR-reverse blot hybridization assay in respiratory specimens for rapid detection and differentiation of mycobacteria in HIV-negative population.

BACKGROUND: Rapid identification of pathogenic Mycobacterium species is critical for a successful treatment. However, traditional method is time-consuming and cannot discriminate isolated non-tuberculosis mycobacteria (NTM) at species level. In the retrospective study, we evaluated the clinical applicability of PCR-reverse blot hybridization assay (PCR-REBA Myco-ID) with clinical specimens for rapid detection and differentiation of mycobacterial species. METHODS: A total of 334 sputum and 362 bronchial alveolar lavage fluids (BALF) from 696 patients with mycobacterium pulmonary disease (MPD) and 210 patients with non-mycobacterium pulmonary disease used as controls were analyzed. Sputum or BALF were obtained for MGIT 960-TBc ID test and PCR-REBA Myco-ID assay. High resolution melt analysis (HRM) was used to resolve inconsistent results of MGIT 960-TBc ID test and PCR-REBA Myco-ID assay. RESULTS: A total of 334 sputum and 362 BALF specimens from 696 MPD patients (292 MTB and 404 NTM) were eventually analyzed. In total, 292 MTBC and 436 NTM isolates (mixed infection of two species in 32 specimens) across 10 Mycobacterium species were identified. The most frequently isolated NTM species were M. intracellulare (n = 236, 54.1%), followed by M. abscessus (n = 106, 24.3%), M. kansasii (n = 46, 10.6%), M. avium (n = 36, 8.3%). Twenty-two cases had M. intracellulare and M. abscessus mixed infection and ten cases had M. avium and M. abscessus mixed infection. A high level of agreement (n = 696; 94.5%) was found between MGIT 960-TBc ID and PCR-REBA Myco-ID (k = 0.845, P = 0.000). PCR-REBA Myco-ID assay had higher AUC for both MTBC and NTM than MGIT 960-TBc ID test. CONCLUSION: PCR-REBA Myco-ID has the advantages of rapid, comparatively easy to perform, relatively low cost and superior accuracy in mycobacterial species identification compared with MGIT 960-TBc ID. We recommend it into workflow of mycobacterial laboratories especially in source-limited countries.

Genetic profiling of CAH Egyptian children: rapid guide to clinical interpretation of common mutations.

OBJECTIVES: The prevalence of CAH in Egypt is reported to be ten times more than that of the worldwide prevalence. The study aimed at genetic screening of children diagnosed with 21-alpha hydroxylase deficiency congenital adrenal hyperplasia (21OHD-CAH). In addition, the study offers a rapid and easy guide for clinical reporting of common mutations for endocrinologists. METHODS: A cohort of 174 unrelated Egyptian children with 21OHD-CAH were screened for 11 common CYP21A2 gene mutations using a strip hybridization assay, and then, bioinformatics analysis was done to report the pathogenicity of the common mutations for clinical classification. RESULTS: The most common mutations were I2 splice and p.Q318X. Deletions/conversions comprised 45.9% of the cohort, whereas 7.4% of the cases were negative for all mutations. The least positively detected point mutations were p.P453S, cluster E6, p.R483P, and p.L307FS, which were detected in fewer than 5% of cases. CONCLUSION: Strip hybridization assay is a rapid screening tool for the diagnosis of CAH. The authors hypothesized an easy and rapid scheme for clinical interpretation of the strip results to gain the highest value of the strip in diagnosis.

Efficient Early Diagnosis of Sepsis Using Whole-Blood PCR-Reverse Blot Hybridization Assay Depending on Serum Procalcitonin Levels.

Sepsis is one of the medical emergencies, and its early detection, within the first hours of development, and proper management improve outcomes. Molecular diagnostic assays using whole blood collected from patients with suspected sepsis have been developed, but the decision making is difficult because of the possibility of false positives, due to contamination. Here, we evaluated the performance of the reverse blot hybridization assay (REBA) Sepsis-ID test for the detection of sepsis-causing microorganisms using whole-blood samples. In addition, the concentrations of C-reactive protein (CRP) and procalcitonin (PCT) were determined to evaluate whether these biomarkers can provide criteria for performing REBA Sepsis-ID in clinical settings. For this study, EDTA-anticoagulated whole blood was simultaneously collected for REBA Sepsis-ID and blood culture from 440 patients with suspected sepsis, from January to October 2015. In addition, CRP and PCT concentrations were measured in 227 patients. The overall positive rates of REBA Sepsis-ID and blood culture were 16.6% (73/440) and 13.9% (61/440), respectively. The pathogen-positive rates of REBA Sepsis-ID and blood culture were 9.8% (43/440) and 9.5% (42/440), respectively. The areas under the receiver operating characteristic (AUROC) curves of PCT and CRP for predicting pathogen-positive results of REBA Sepsis-ID were 0.72 and 0.69, respectively. The PCT concentrations in the group of patients aged ≥50 years were significantly higher than those in the group aged <50 years. After adjusting for age, the PCT AUROC value was 0.77 for predicting pathogen-positive results of REBA Sepsis-ID. The optimal cutoff values of PCT concentrations for subsequent application of REBA Sepsis-ID were 0.12 ng/mL in all patients and 0.22 ng/mL in patients aged ≥50 years. Our observations showed that REBA Sepsis-ID using whole blood was advantageous for the early detection of sepsis-causing microorganisms, and the PCT concentration could be used to determine the necessity of using REBA Sepsis-ID in clinical settings. The application of REBA Sepsis-ID using whole blood, based on the PCT concentration, may contribute to a highly efficient detection of sepsis-causing microorganisms.

Electrochemical biosensor for miRNA-21 based on gold-platinum bimetallic nanoparticles coated 3-aminopropyltriethoxy silane.

We report an electrochemical biosensor based on gold platinum bimetallic nanoparticles (AuPtBNPs)/3-aminopropyltriethoxy silane (APTS) nanocomposite coated fluorine-doped tin oxide (FTO) as a biosensing platform for hybridization-based detection of miRNA-21. Field Emission-Scanning Electron Microscopy (FE-SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and electrochemical measurements were carried out to ensure the successful construction of the biosensor. The amount of cDNA immobilized on electrode surface and hybridization time required for the miRNA-21 sensing were optimized. The biosensing platform showed detection limit of 0.63 fM with wide linear range i.e. 1 fM-100 nM for miRNA-21 detection. The biosensing strategy demonstrates a good recovery yield from 90.18% to 94.6% in serum samples. It offers good selectivity for its complementary miRNA compared to the non-complementary miRNAs. Other analytical features of the biosensor such as stability, reusability and reproducibility were also tested, providing appropriate results.

A Rapid and Accurate Detection Approach for Multidrug-Resistant Tuberculosis Based on PCR-ELISA Microplate Hybridization Assay.

Rapid and accurate diagnosis of multidrug-resistant tuberculosis (MDR-TB) is important for timely and appropriate therapy. In this study, a rapid and easy-to-perform molecular test that integrated polymerase chain reaction (PCR) amplification and a specific 96-well microplate hybridization assay, called PCR-ELISA (enzyme-linked immunosorbent assay), were developed for detection of mutations in rpoB, katG, and inhA genes responsible for rifampin (RIF) and isoniazid (INH) resistance and prediction of drug susceptibility in Mycobacterium tuberculosis clinical isolates. We evaluated the utility of this method by using 32 multidrug-resistent (MDR) isolates and 22 susceptible isolates; subsequently, we compared the results with data obtained by conventional drug susceptibility testing and DNA sequencing. The sensitivity and specificity of the PCR-ELISA test were 93.7% and 100% for detecting RIF resistance, and 87.5% and 100% for detecting INH resistance, respectively. These results were comparable to those yielded by commercially available molecular tests such as the GenoType MTBDRplus assay. Based on the aforementioned results, we conclude that the PCR-ELISA microplate hybridization assay is a rapid, inexpensive, convenient, and reliable test that will be useful for rapid diagnosis of MDR-TB, for improved clinical care.

Magnetosomes for bioassays by merging fluorescent liposomes and magnetic nanoparticles: encapsulation and bilayer insertion strategies.

Magnetized liposome (magnetosomes) labels can overcome diffusion limitations in bioassays through fast and easy magnetic attraction. Our aim therefore was to advance the understanding of factors influencing their synthesis focusing on encapsulation strategies and synthesis parameters. Magnetosome synthesis is governed by the surface chemistry and the size of the magnetic nanoparticles used. We therefore studied the two possible magnetic labelling strategies, which are the incorporation of small, hydrophobic magnetic nanoparticles (MNPs) into the bilayer core (b-liposomes) and the entrapment of larger hydrophilic MNPs into the liposomes' inner cavity (i-liposomes). Furthermore, they were optimized and compared for application in a DNA bioassay. The major obstacles observed for each of these strategies were on the one hand the need for highly concentrated hydrophilic MNPs, which is limited by their colloidal stability and costs, and on the other hand the balancing of magnetic strength vs. size for the hydrophobic MNPs. In the end, both strategies yielded magnetosomes with good performance, which improved the limit of detection of a non-magnetic DNA hybridization assay by a factor of 3-8-fold. Here, i-liposomes with a magnetization yield of 5% could be further improved through a simple magnetic pre-concentration step and provided in the end an 8-fold improvement of the limit of detection compared with non-magnetic conditions. In the case of b-liposomes, Janus-like particles were generated during the synthesis and yielded a fraction of 15% magnetosomes directly. Surprisingly, further magnetic pre-concentration did not improve their bioassay performance. It is thus assumed that magnetosomes pull normal liposomes through the magnetic field towards the surface and the presence of more magnetosomes is not needed. The overall stability of magnetosomes during storage and magnetic action, their superior bioassay performance, and their adaptability towards size and surface chemistry of MNPs makes them highly valuable signal enhancers in bioanalysis and potential tools for bioseparations. Graphical abstract.

Solid-Phase Hybridization Assay for Detection of Mutated Cancer DNA by Fluorescence.

We report a straightforward protocol for the detection of mutated DNA extracted from cancer cells. The assay combines a step-wise solid-phase hybridization and a readout by fluorescence emission. We detect a single-nucleotide polymorphism in two human oncogenes, BRAF and EGFR, and reach a limit of the detection of 300 pM by conventional fluorometry. The protocol described herein may be used as a foundation for development of automatic optimized assays capable for detection of mutant DNA and RNA in vitro and in cells.