Methods for rapid detection of infectious bacteria and antimicrobial-resistant pathogens have evo... more Methods for rapid detection of infectious bacteria and antimicrobial-resistant pathogens have evolved significantly over the last decade. Many of the new procedures are nucleic acid-based and replace conventional diagnostic methods like culturing which is time consuming especially with fastidious and slow growing microorganisms. The widespread use of antibiotics has resulted in an increased number of cases with resistant microorganisms such as methicillin-resistant Staphylococcus aureus, vancomycin resistant enterococci, and multidrug-resistant Mycobacterium tuberculosis. Rapid detection of these pathogens is important to isolate patients and prevent further spreading of the diseases. Newly developed diagnostic procedures are superior with respect to turnaround time, sensitivity and specificity. Methods like multiplex real time PCR and different array-based technologies offer the possibility of multiparameter assays where several pathogens and antibiotic resistance genes can be detected simultaneously.
Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested... more Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-aminop...
BACKGROUND: A rapid microtiter plate based sandwich hybridization assay was developed for detecti... more BACKGROUND: A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules. RESULTS: The sensitivity of the assay was approximately 1.2 x 109 (2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities. CONCLUSIONS: The here presented sandwich hybridisation method was suc...
... EXPRESSION ANALYSIS BY LNA ENHANCED MICROARRAYS Rolf Søkilde 1, 2, Bogumil Kaczkowski 2, Kim ... more ... EXPRESSION ANALYSIS BY LNA ENHANCED MICROARRAYS Rolf Søkilde 1, 2, Bogumil Kaczkowski 2, Kim Bundvig Barken 2, Peter Mouritzen 2, Søren ... Therefore, one can also look at how many miRNAs have higher fold change then a certain threshold, in this case: 2 (see ...
Community structure and co-operation in biofilms The study of biofilm considers the close associa... more Community structure and co-operation in biofilms The study of biofilm considers the close association of micro-organisms with each other at interfaces and the consequent physiological adaptation to the proximity of other cells and surfaces. As such it is relevant to a variety of disciplines, ...
Methods for rapid detection of infectious bacteria and antimicrobial-resistant pathogens have evo... more Methods for rapid detection of infectious bacteria and antimicrobial-resistant pathogens have evolved significantly over the last decade. Many of the new procedures are nucleic acid-based and replace conventional diagnostic methods like culturing which is time consuming especially with fastidious and slow growing microorganisms. The widespread use of antibiotics has resulted in an increased number of cases with resistant microorganisms such as methicillin-resistant Staphylococcus aureus, vancomycin resistant enterococci, and multidrug-resistant Mycobacterium tuberculosis. Rapid detection of these pathogens is important to isolate patients and prevent further spreading of the diseases. Newly developed diagnostic procedures are superior with respect to turnaround time, sensitivity and specificity. Methods like multiplex real time PCR and different array-based technologies offer the possibility of multiparameter assays where several pathogens and antibiotic resistance genes can be detected simultaneously.
Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested... more Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-aminop...
BACKGROUND: A rapid microtiter plate based sandwich hybridization assay was developed for detecti... more BACKGROUND: A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules. RESULTS: The sensitivity of the assay was approximately 1.2 x 109 (2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities. CONCLUSIONS: The here presented sandwich hybridisation method was suc...
... EXPRESSION ANALYSIS BY LNA ENHANCED MICROARRAYS Rolf Søkilde 1, 2, Bogumil Kaczkowski 2, Kim ... more ... EXPRESSION ANALYSIS BY LNA ENHANCED MICROARRAYS Rolf Søkilde 1, 2, Bogumil Kaczkowski 2, Kim Bundvig Barken 2, Peter Mouritzen 2, Søren ... Therefore, one can also look at how many miRNAs have higher fold change then a certain threshold, in this case: 2 (see ...
Community structure and co-operation in biofilms The study of biofilm considers the close associa... more Community structure and co-operation in biofilms The study of biofilm considers the close association of micro-organisms with each other at interfaces and the consequent physiological adaptation to the proximity of other cells and surfaces. As such it is relevant to a variety of disciplines, ...
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