Biomedical subjects

Knut Rudi

Publications and source records attributed to Knut Rudi.

30 records · Page 2Linked to original sources

PubMed2004

Potential influence of the first PCR cycles in real-time comparative gene quantifications.

There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.

Artifacts↗

PubMed2003

Real-time closed tube single nucleotide polymorphism (SNP) quantification in pooled samples by quencher extension (QEXT).

Quencher extension (QEXT) is a novel single step closed tube real-time method to quantify SNPs using reporters and quenchers in combination with primer extension. A probe with a 5'-reporter dye is single base extended with a dideoxy nucleotide containing a quencher dye if the target SNP allele is present. The extension is recorded from the quenching (reduced fluorescence) of the reporter dye. This avoids the influence of the unincorporated dye-labeled nucleotides, resulting in high accuracy and a high signal-to-noise ratio. The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermo-cycling reaction. We tested the QEXT assay using five SNPs in the Listeria monocytogenes inlA gene as a model system. The presence of the target SNP alleles was determined with high statistical confidence (P < 0.0005). The quantitative detection limits were between 0 and 5% for the targeted SNP alleles on a background of other SNP alleles (P < 0.05). The QEXT method is directly adaptable to current real-time PCR equipment and is thus suited for high throughput and a wide application range.

DNA Probes↗

PubMed2003

Protocols for 16S rDNA array analyses of microbial communities by sequence-specific labeling of DNA probes.

Conserved Sequence↗

PubMed2003

A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed.

We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5' 'HEAD' region and a 3' region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5' nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.

Animal Feed↗

PubMed2003

Multi locus fingerprinting of Listeria monocytogenes by sequence-specific labeling of DNA probes combined with array hybridization.

We have developed an alternative multi locus sequence typing (MLST) approach that targets the variable genetic changes directly in a DNA array format. Our approach is based on DNA array hybridization in combination with sequence-specific labeling of oligonucleotide probes. Listeria monocytogenes was chosen for the development and evaluation of the assay. The genes hlyA, iap, flaA, inlA and actA were targeted. Twenty-nine suitable probe regions were identified within these genes. The DNA array results from 32 different strains were compared to serotype and amplified fragment length polymorphism data. This comparison showed that our DNA array method gave good discrimination between the strains analyzed. In conclusion, the DNA array-based MLST method is a promising tool for fingerprint bacteria.

Bacterial Typing Techniques↗

PubMed2003

Comparison of membranes and glass slides for 16S rDNA array analyses of microbial communities by sequence-specific labeling of DNA probes.

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for a DNA array based analyzes of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions, and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Arrays prepared on positively charged nylon membranes and coated glass slides were compared. The advantage of using membranes is that chromogenic signal amplification can be used for the detection. Furthermore, the chromogenic detection does not require any sophisticated equipment. The advantage of the glass slides is that multiple fluorescence colors can be detected simultaneously, and that internal controls can be used for normalization. This approach is also suited for high throughput screenings.

Bacteria↗

PubMed2003

Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments.

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.

Animals↗

PubMed2003

Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains.

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.

Amino Acid Sequence↗

PubMed2003

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR.

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.

Bacteria↗

PubMed2002

Development and application of new nucleic acid-based technologies for microbial community analyses in foods.

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.

Bacteria↗

PubMed2002

Development and evaluation of a 16S ribosomal DNA array-based approach for describing complex microbial communities in ready-to-eat vegetable salads packed in a modified atmosphere.

There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets 16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities. Pseudomonas spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4 degrees C. The Pseudomonas population also dominated the batch containing Norwegian lettuce after storage at 10 degrees C. On the contrary, Enterobacteriaceae and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10 degrees C. In that batch, the Enterobacteriaceae also were abundant after storage at 4 degrees C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions.

Bacteria↗

PubMed2002

Nested evolution of a tRNA(Leu)(UAA) group I intron by both horizontal intron transfer and recombination of the entire tRNA locus.

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA(Leu)(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA(Ile)(CAU) and tRNA(Arg)(CCU) genes in alpha- and beta-proteobacteria, respectively, than with other cyanobacterial tRNA(Leu)(UAA) group I introns. The divergent tRNA(Leu)(UAA) intron is sporadically distributed both within the Nostoc and the Microcystis radiations. The complete tRNA gene, including flanking regions and intron from Microcystis aeruginosa strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the Microcystis genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA(Leu)(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA(Leu)(UAA) gene which is located within a unstable genome region.

Base Sequence↗