| 1. |
- Bjerketorp, J., et al.
(författare)
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Rapid lab-on-a-chip profiling of human gut bacteria
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 72:1, s. 82-90
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Tidskriftsartikel (refereegranskat)abstract
- The human gut microbiota has a substantial impact on human health. Different factors such as disease, diet and drug use can have significant impacts on the gut microbiota. Therefore, it is of interest to have simple, rapid methods for analysis of the composition of the gut microbiota for clinical diagnostic purposes. Since only a minor fraction of the gastrointestinal bacterial community is presently possible to cultivate, molecular approaches are currently the best suited to investigate its composition. However, most of these molecular approaches require technical expertise and expensive equipment to run and they are not routinely available. Ideally, the analyses should be point-of-care options that can be run on a chip. In this study, an existing lab-on-chip (LOC) system for sizing/quantifying DNA was combined with length heterogeneity PCR (LH-PCR), a PCR-based profiling method targeting bacterial 16S rRNA gene sequences, to develop a fast, straightforward, reproducible, and economical method for profiling bacterial communities. The LOC LH-PCR method was first evaluated using a standardized gut cocktail containing genomic DNA from eight different bacterial species representing different genera of relevance for human health. The method was also tested on DNA that was directly extracted from human faecal samples and it was consistently capable of detecting alterations in the bacterial samples before and after antibiotic treatment. Although the resolution of the method needs improvement, this study represents the first step towards development of a diagnostic LOC for profiling gut bacterial communities.
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| 2. |
- Crocetti, Greg, et al.
(författare)
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An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence in situ hybridisation (FISH)
- 2006
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 65:1, s. 194-201
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescence in situ hybridisation (FISH) is a common and popular method used to investigate microbial populations in natural and engineered environments. DNA oligonucleoticle probes require accurate determination of the optimal experimental conditions for their use in FISH Oligonucleotides targeting the rRNA of methanogenic Archaea at various taxonomic levels have previously been published, although when applied in FISH, no optimisation data has been presented In this study, 3000 Euryarchaeota 16S rRNA gene sequences were phylogenetically analysed and previously published oligonucleoticles were evaluated for target group accuracy. Where necessary, modifications were introduced or new probes were designed. The updated set of probes was optimised for use in FISH for a more accurate detection of methanogenic Archaea. (c) 2005 Elsevier B.V. All rights reserved.
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| 3. |
- Eriksson, Ronnie, et al.
(författare)
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Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout
- 2009
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 78:2, s. 195-202
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Tidskriftsartikel (refereegranskat)abstract
- A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.
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| 4. |
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| 5. |
- Fellström, Claes, et al.
(författare)
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Identification and genetic fingerprinting of Brachyspira species
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 72:2, s. 133-140
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Tidskriftsartikel (refereegranskat)abstract
- Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intennedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.
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| 6. |
- Ferrando, R, et al.
(författare)
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3-Hydroxy fatty acids in saliva as diagnostic markers in chronic periodontitis
- 2005
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 62:3, s. 285-291
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Tidskriftsartikel (refereegranskat)abstract
- Saturated straight- and branched-chain 3-hydroxy fatty acids (3-OH FAs) of 10-18 carbon chain lengths were determined in saliva from 27 individuals with chronic periodontitis and 18 healthy individuals by using gas chromatography-tandem mass spectrometry. Of the 14 different 3-OH FAs detected, 3-OH-C-i17:0 was the most abundant in the periodontitis samples while 3-OH-C-14:0 was the most abundant in the healthy individuals. Considering the relative percentages of 3-OH-C-12:0, 3-OH-C-14:0, 3OH-C-i17:0, and 3-OH-C-17:0, 95.6% of all cases were correctly classified as healthy individuals or periodontitis patients by means of discriminant analysis. The sensitivity, specificity, positive predictive value and negative predictive value of 3-OH FA analysis in diagnosing peridontitis were, respectively, 0.92, 1.00, 1.00, and 0.90. The results indicate that 3-OH FA analysis of saliva samples is a useful diagnostic method in chronic periodontitis.
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| 7. |
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| 8. |
- Ihalin, R, et al.
(författare)
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16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains.
- 2006
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 65:3, s. 417-424
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Tidskriftsartikel (refereegranskat)abstract
- Certain serotypes of Actinobacillus actinomycetemcomitans seem to prefer coexistence in vivo. The 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was tested for its capability to distinguish coexisting A. actinomycetemcomitans strains of different serotypes or genetic lineages and to determine their proportions in vitro. The migration pattern of the PCR amplicon from serotype c differed from those of the other serotypes. Contrary to the strains of serotypes c, d, and e, strains of serotypes a, b, and f consistently demonstrated intra-serotype migration patterns similar to each other. Since the migration patterns differed between serotype c and b strains a strain of each was used to determine their proportional representation in a strain mixture. The strains were distinguishable from each other above the 5% PCR-DGGE detection level (12.5 ng DNA/1.5 x 10(6) cells). DGGE provides a promising tool for in vitro studies on the coexistence of different genetic lineages of A. actinomycetemcomitans.
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| 9. |
- Karched, M, et al.
(författare)
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A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans
- 2007
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 68:1, s. 46-51
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Tidskriftsartikel (populärvet., debatt m.m.)abstract
- Tenacious adherence and autoaggregation of wild-type Actinobacillus actinomycetemcomitans strains jeopardize reliability of determined cell concentrations, e.g. for studies on bacteria-host interactions. We first compared the efficacy of two methods, an indirect and a direct method, for homogenizing cell suspensions of a wild-type, autoaggregating (SA269) strain and of a non-autoaggregating laboratory variant (ATCC 43718) used as a reference. Since the direct method left visible clumps in SA269 suspension, only the indirect method was further tested. In serial dilutions of the homogenized cell suspensions of strains SA269 and ATCC 43718, the OD(600) values (R(2)=0.99, R(2)=0.99, respectively) and protein concentrations (R(2)=0.93, R(2)=0.95, respectively) correlated significantly (all P<0.002) with the dilution factor. There were no differences (P>0.05) in the bacterial viable counts between the two strains or between suspending solutions, i.e., PBS and water, the cell concentrations demonstrating 1x10(9) cells/ml at OD(600)=1. Repeated microscopic cell counts did not differ (P>0.05) from each other. Large aggregates occurred as 1% of cell units counted. Dispersing bacterial mass indirectly to solution leads to homogeneous cell suspensions with repeatable cell concentrations. Viability of A. actinomycetemcomitans was also maintained when cells were suspended in water.
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| 10. |
- Lindstedt, Bjørn-Arne, et al.
(författare)
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Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 72:2, s. 141-148
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Tidskriftsartikel (refereegranskat)abstract
- The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).
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| 11. |
- Liu, Yanling, 1974-, et al.
(författare)
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Confirmative electric DNA array-based test for food poisoning Bacillus cereus
- 2007
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 70:1, s. 55-64
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Tidskriftsartikel (refereegranskat)abstract
- Detection of the full set of toxin encoding genes involved in gastrointestinal diseases caused by B. cereus was performed. Eight genes determining the B. cereus pathogenicity, which results in diarrhea or emesis, were simultaneously evaluated on a 16-position electrical chip microarray. The DNA analyte preparation procedure comprising first 5 min of ultrasonic treatment, DNA extraction, and afterwards an additional 10 min sonication, was established as the most effective way of sample processing. No DNA amplification step prior to the analysis was included. The programmed assay was carried out within 30 min, once the DNA analyte from 10(8) bacterial cells, corresponding to one agar colony, was subjected to the assay. In general, this work represents a mature analytical way for DNA review. It can be used under conditions that require almost immediate results.
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| 12. |
- Lundén, K., et al.
(författare)
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Heterologous array analysis in Heterobasidion : Hybridisation of cDNA arrays with probe from mycelium of S, P or F-types
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 75:2, s. 219-224
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Tidskriftsartikel (refereegranskat)abstract
- Because of the close relatedness between three species of Heterobasidion annosum (P type), Heterobasidion parviporum (S-type) and Heterobasidion abietinum (F-type). we investigated the possible use of arrays from one species for studies of gene expression in the other. Clones containing partial cDNAs from 94 identifiable genes expressed during spore germination and differentiation in H. parviporum were printed manually in six replications on nylon membranes. The membrane was hybridized with chemifluorescent labelled cDNA from actively growing mycelia of H. parviporum, H. annosum or H. abietinum, cultivated on a non-selective substrate. Product-moment correlation coefficient varied between 0.81 and 0.49. Due to the level of correlation, in the gene expression among the intersterility groups, we concluded that the cDNA array of one can be used to study gene expression in the others.
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| 13. |
- Monstein, Hans-Jurg, 1946-, et al.
(författare)
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Multiple displacement amplification of DNA from human colon and rectum biopsies: Bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis
- 2005
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 63:3, s. 239-247
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Tidskriftsartikel (refereegranskat)abstract
- Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens. (c) 2005 Elsevier B.V. All rights reserved.
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| 14. |
- Riazi, Shadi, et al.
(författare)
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Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides
- 2007
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 71:1, s. 87-89
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Tidskriftsartikel (refereegranskat)abstract
- BioRad's Rotofor (R) system has been frequently used for the purification of proteins and smaller pepticles such as bacteriocins. In this study, we report that some commercially available ampholytes used with the Rotofor (R) isoelectric focusing system possess antimicrobial activity, which may interfere with the purification of bacteriocins and bacteriocin-like substances.
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| 15. |
- Storm, Martin, et al.
(författare)
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Comparison of real-time PCR and pyrosequencing for typing Bordetella pertussis toxin subunit 1 variants
- 2006
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 65:1, s. 153-158
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Tidskriftsartikel (refereegranskat)abstract
- We describe two newly developed methods for rapid typing of the pertussis toxin subunit 1 gene (ptxS1). A real-time PCR assay based on hybridization probes and a Pyrosequencing assay were developed and the specificity, sensitivity, cost, hands-on time and post-assay data processing were compared to Sanger sequencing. Both methods enabled discrimination of all four allelic variants, correctly identified all ptxS1 alleles of 143 strains tested and proved suitable for large-scale screening of B. pertussis strains.
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| 16. |
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| 17. |
- Wolffs, Petra, et al.
(författare)
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Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells
- 2005
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 60:3, s. 315-323
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Tidskriftsartikel (refereegranskat)abstract
- Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized. (C) 2004 Published by Elsevier B.V.
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