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We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 degrees C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in < 20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics.
The hydrolytic degradation and corresponding content release of capsules made of poly(d,l-lactic-co-glycolic acid) (PLGA) strongly depends on the composition and material properties of the initially applied copolymer. Consecutive or simultaneous release from capsule batches of combinable material compositions, therefore, offers high control over the bioavailability of an encapsulated drug. The keynote of this study was the creation of a superordinated database that addressed the correlation between the release kinetics of filling agents with different molecular weights from PLGA capsules of alternating composition. Fluorescein isothiocyanate (FITC)-dextran (with molecular weights of 4, 40, and 2000 kDa) was chosen as a model analyte, whereas the copolymers were taken from various 50:50 PLGA, 75:25 PLGA, and polylactide blends. With reference to recent publications, the capsule properties, such as the size, morphology, and encapsulation efficiency, were further modified during production. Hence, uniform microdisperse and polydisperse submicrometer nanocapsules were prepared by two different water-in-oil-in-water emulsification techniques, and additional effects on the size and morphology were achieved by capsule solidification in two different sodium salt buffers. The qualitative and quantitative examination of the physical capsule properties was performed by confocal laser scanning microscopy, scanning electron microscopy, and Coulter counting techniques to evaluate the capsule size distribution and the morphological appearance of the different batches. The corresponding agent release was quantified by fluorescence measurement of the FITC-dextran in the incubation media and by the direct measurement of the capsule brightness via fluorescence microscopy. In summary, the observed agent release showed a highly controllable flexibility depending on the PLGA blends, preparation methods, and molecular weight of the used filling substances. (c) 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 4219-4228, 2013
Background: Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures.
Results: Four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity. We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168.
Conclusions: The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.zeige weniger
The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.
An artificial gene was constructed combining the T7 promoter and terminator with the EGFP-gene from the plasmid pEGFP. The functionality of the construct was shown by in vitro translation. The gene-construct was immobilised on a planar glass surface. The transcription was performed on the immobilised gene and mRNA was determined by RT-PCR. Multiple use of the immobilised gene was demonstrated
Microarrays are new analytical devices that allow the parallel and simultaneous detection of thousands of target compounds. Microarrays, also called DNA chips, are widely used in gene expression, the genotyping of individuals, point mutations, detection of single nucleotide polymorphisms, and short tandem repeats. Microarrays have highly specific base-pair interactions with labeled complementary strands, which makes this technology to a powerful analytical device for monitoring whole genomes. In this article, we provide a survey of the common microarray manufacturing methods, from the selection of support material to surface structuring, immobilization and hybridization, and finally the detection with labeled complementary strands. Special attention is given to the immobilization of single strands, since fast chemical reactions, the creation of homogeneous surface functionalities as well as an oriented coupling are crucial pre-conditions for a good spot morphology and microarrays of high quality