| 51. |
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| 52. |
- Periyannan Rajeswari, Prem Kumar, et al.
(författare)
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Multiple pathogen biomarker detection using an encoded bead array in droplet PCR
- 2017
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 139, s. 22-28
-
Tidskriftsartikel (refereegranskat)abstract
- We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color coded Luminex beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.
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| 53. |
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| 54. |
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| 55. |
- 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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| 56. |
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| 57. |
- Ryberg, Anna, et al.
(författare)
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Comparison of (GTG)(5)-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 84:2, s. 183-188
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Tidskriftsartikel (refereegranskat)abstract
- Molecular typing of Klebsiella species has become important for monitoring dissemination of beta-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)(5)- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19K pneumoniae (some with an ESBL-phenotype). 35 K. oxytoca isolates, five K pneumoniae, two K oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)(5) and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)(5) and ITS-PCR analysis revealed that K pneumoniae and K oxytoca isolates. reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)(5) and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates. (C) 2010 Elsevier B.V. All rights reserved.
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| 58. |
- Sannö, Axel, et al.
(författare)
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The development of a screening protocol for Salmonella spp. and enteropathogenic Yersinia spp. in samples from wild boar (Sus scrofa) also generating MLVA-data for Y. enterocolitica and Y. pseudotuberculosis
- 2018
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 150, s. 32-38
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Tidskriftsartikel (refereegranskat)abstract
- Salmonellosis and yersiniosis are notifiable human diseases that are commonly associated with contaminated food. Domestic pigs as well as wild boars and other wild-life have been identified as reservoirs of these bacteria. Methods for cultivation and molecular epidemiological investigations of Salmonella spp. are well established, however, cultivation of enteropathogenic Yersinia spp. is time- consuming and the commonly used method for molecular epidemiological investigations, pulsed-field gel electrophoresis, lack in discriminatory power. The aim of this study was to develop and evaluate a screening protocol well suited for wildlife samples and other highly contaminated samples. The method is based on PCR-screening followed by Multiple Loci Variant number tandem repeat Analysis (MLVA) on enrichment broth to obtain molecular epidemiological data for enteropathogenic Yersinia spp. without the need for pure isolates. The performance of the protocol was evaluated using wild boar samples (n = 354) including tonsils, faeces and lymph nodes from 90 Swedish wild boars. The new protocol performed as well as or better than the established ISO-standards for detection and cultivation of Y. enterocolitica and Salmonella spp., however for cultivation of Y. pseudotuberculosis, further development is needed. The selection for motility seems beneficial for the enrichment of Salmonella spp. and Y. enterocolitica. Further, the selective enrichment prior to PCR-analysis eliminates inhibitory factors present in the original sample. In total, ten isolates of Y. enterocolitica of various bio-serotypes were obtained, and the MLVA-profile of these isolates were consistent with the profiles from the corresponding enrichment broth. Further, 22 isolates of Salmonella spp. comprising six different serovars were obtained with S. Fulica, S. Hadar and a monophasic S. Typhimurium being the most common. In conclusion, the presented screening protocol offers a rapid and efficient way to obtain prevalence data from a large sample set as well as MLVA-data within a short time frame. These results can hence improve the knowledge on the epidemiology and distribution of these pathogens and their importance to public health.
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| 59. |
- Sjöberg, Fei, et al.
(författare)
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Are all faecal bacteria detected with equal efficiency? A study using next-generation sequencing and quantitative culture of infants' faecal samples
- 2020
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 177
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Many species of intestinal bacteria are present in moderate numbers in the faecal microbiota, which is dominated by obligate anaerobes. Little is known regarding the detection sensitivity of next-generation sequencing for these microbes in samples of complex microbiota. Methods: Twenty stool samples from six healthy infants, who were followed from 1 week to 1 year of age, were previously cultured quantitatively for total population counts, as well as for counts of relevant facultative bacteria and a limited selection of obligate anaerobes that are prevalent in the neonatal microbiota. The same samples were analysed by Next-generation sequencing (NGS, pyrosequencing) of the 16S rRNA gene (V1–V3 regions; average read length, 500 nucleotides; average number of reads per sample, 30,000). We used the bacterial culture data to determine the lowest bacterial populations that could be detected by NGS. Different DNA extraction kits (QIAamp DNA Stool Mini, ZR Faecal DNA MiniPrep, and PowerSoil DNA Isolation) were compared for efficacy in extracting DNA from Gram-negative and Gram-positive Type strains. Results: NGS yielded one read per 106 CFU/g faeces of the Gram-negative commensal gut bacteria Bacteroides and Enterobacteriaceae, but only one read per 108 CFU/g faeces of Gram-positive bifidobacteria. The Gram-positive facultative bacteria Enterococcus was often undetectable by DNA-based methods despite being present at >106 CFU/g faeces. The DNA extraction kits tested varied considerably in their ability to extract DNA from bacterial samples, and showed considerably lower efficacies in extracting DNA from Gram-positive than from Gram-negative bacteria. Conclusions: NGS has lower sensitivity for detecting Gram-positive bacteria than Gram-negative bacteria, due at least in part to inefficient extraction of DNA from Gram-positive bacteria. Therefore, enzymatic lysis may enhance the yield of DNA and increase the sensitivity of NGS methods for Gram-positive bacteria, and the inclusion of positive and negative controls during DNA extraction is indicated for validation purposes. The differential extraction of DNA from bacterial samples by different DNA extraction kits may limit comparability between studies on the gut microbiota. Finally, quantitative culture methods detect certain bacteria with greater sensitivity than NGS techniques, and thus culture- and DNA-based methods can be used in tandem to define the complex composition of the gut microbiota with greater accuracy.
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| 60. |
- Sjöberg, Fei, et al.
(författare)
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Comparison between terminal-restriction fragment length polymorphism (T-RFLP) and quantitative culture for analysis of infants' gut microbiota
- 2013
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 94:1, s. 37-46
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Tidskriftsartikel (refereegranskat)abstract
- The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism (T-RFLP). A database was constructed for direct identification of T-RFLP peaks by analysis of pure-culture bacteria and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at >10(6) CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts. In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be identified using the database. T-RFLP readily identified bacteria to the genus level and also provided some sub-genus discrimination. Both T-RFLP and culture identified Bifidobacterium, Clostridium and Bacteroides spp. among the most common colonizers of the infantile microbiota throughout the first year of life. T-RFLP analysis showed that microbiota complexity was high in the first weeks of life, declined to a minimum at 1-2 months of age, and thereafter increased again. Principal component analysis revealed that early samples (1 week-6 months) chiefly differed between individual infants, while 12-month samples were similar between children, but different from the early samples. Our results indicate that T-RFLP has high sensitivity and adequate taxonomic discrimination capacity for analysis of gut microbiota composition, but that both culture and molecular based analysis have limitations and both approaches may be needed to obtain a full picture of the complex gut microbiota.
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| 61. |
- 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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| 62. |
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| 63. |
- Sune, Dan, et al.
(författare)
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Optimization of 16S rRNA gene analysis for use in the diagnostic clinical microbiology service
- 2020
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Ingår i: Journal of Microbiological Methods. - : ELSEVIER. - 0167-7012 .- 1872-8359. ; 170
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Tidskriftsartikel (refereegranskat)abstract
- Broad-range amplification and sequencing of the 16S rRNA gene, directly from clinical samples, is a method that potentially allows detection of any cultivable or non-cultivable bacteria. However, the method is prone to false positive results due to PCR contamination. Another concern is the human DNA abundance compared to bacterial DNA in samples from sterile sites. Those factors may decrease the sensitivity and specificity of the assay and can complicate the analysis and interpretation of the results. The objective of this prospective study was to try to avoid the most common pitfalls, mentioned above, and develop a molecular 16S assay with a high clinical sensitivity and specificity. Fifty-six consecutive tissue samples from patients with suspected deep infections were extracted by 3 different DNA-extraction methods; two based on a principle of bacterial DNA enrichment, and one conventional DNA extraction method. We compared three primer pairs, including both conventional and DPO principle, targeting different variable regions of the 16S rRNA gene. Results from routine tissue culture were used as reference. Clinical data was recorded from patient charts and analyzed in parallel. Of a total of 56 samples, collected from 39 patients, 70% (39 samples) were assessed as true infections by analysis of clinical data. Bacterial enrichment extraction increased sensitivity from 54% to 72%. The 2 sets of primer pairs defining region V1-V3 and V3-V4, showed similar sensitivity, but DPO-primers resulted in better specificity, i.e. less contaminations. The primer pairs covering V1-V8 show significantly lower sensitivity (p amp;lt; .001) than V1-V3 and V3-V4. Optimizing extraction protocols and choice of primers can increase the sensitivity and specificity of a molecular 16S-analysis, rendering a valuable complement to tissue culture.
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| 64. |
- Svensson, Erik, 1959, et al.
(författare)
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Quantitative analyses of mycobacteria in water: adapting methods in clinical laboratories.
- 2011
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Ingår i: Journal of microbiological methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 87:1, s. 114-5
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Tidskriftsartikel (refereegranskat)abstract
- An outbreak of occupational hot tub lung necessitated quantitative analysis of mycobacteria in water samples. We combined procedures for cultivation of mycobacteria in urine and quantitative analyses of dialysis water. Whirlpool spa water samples were analyzed showing promising results. In conclusion, quantitative mycobacterial culture of water is possible by adapting methods routinely used in clinical laboratories.
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| 65. |
- Szponar, Bogumila, et al.
(författare)
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Limitations in the use of 3-hydroxy fatty acid analysis to determine endotoxin in mammalian samples
- 2002
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Ingår i: Journal of Microbiological Methods. - 1872-8359 .- 0167-7012. ; 50:3, s. 283-289
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Tidskriftsartikel (refereegranskat)abstract
- 3-Hydroxy fatty acids (3-OH FAs) of 10-18-carbon chain lengths are constituents of the lipopolysaccharide of Gram-negative bacteria. These acids are used as chemical markers for determining endotoxin in environmental samples. The present communication addresses the question whether this type of analysis also would be applicable to mammalian samples. Low levels (6.1 +/- 1.6-94.0 +/- 23.2 pmol/ml) of the studied 3-OH FAs were detected in blood from both conventional and germ-fine rats. The levels were considerably higher (0.0-1.06 +/- 0.17 nmol/mg) in livers. The amounts of the 3-OH FAs did not differ between the two groups of rats. All analyses were made by gas chromatography-tandem mass spectrometry (GC-MSMS) for unequivocal identification. The results illustrate a limitation in using 3-OH FA analysis to determine endotoxin in mammalian samples since these acids may represent not only endotoxin but also products from mammalian mitochondrial fatty acid beta-oxidation. (C) 2002 Elsevier Science B.V. All rights reserved.
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| 66. |
- Tavares, F, et al.
(författare)
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A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia
- 2000
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Ingår i: Journal of Microbiological Methods. - 0167-7012 .- 1872-8359. ; 39:2, s. 171-178
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Tidskriftsartikel (refereegranskat)abstract
- A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris-HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5+/-7.44 pg protein per extraction procedure from exponentially growing cells, corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50+/-0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied. (C) 2000 Elsevier Science B.V. All rights reserved.
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| 67. |
- Thierry, S., et al.
(författare)
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A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis
- 2013
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 95:3, s. 357-365
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Tidskriftsartikel (refereegranskat)abstract
- Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2. ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis. © 2013 Elsevier B.V.
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| 68. |
- Ungphakorn, Wanchana, et al.
(författare)
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Evaluation of automated time-lapse microscopy for assessment of in vitro activity of antibiotics
- 2017
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 132, s. 69-75
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Tidskriftsartikel (refereegranskat)abstract
- This study aimed to evaluate the potential of a new time-lapse microscopy based method (oCelloScope) to efficiently assess the in vitro antibacterial effects of antibiotics. Two E. con and one P. aeruginosa strain were exposed to ciprofloxacin, colistin, ertapenem and meropenem in 24-h experiments. Background corrected absorption (BCA) derived from the oCelloScope was used to detect bacterial growth. The data obtained with the oCelloScope were compared with those of the automated Bioscreen C method and standard time-kill experiments and a good agreement in results was observed during 6-24 h of experiments. Viable counts obtained at 1, 4, 6 and 24 h during oCelloScope and Bioscreen C experiments were well correlated with the corresponding BCA and optical density (OD) data. Initial antibacterial effects during the first 6 h of experiments were difficult to detect with the automated methods due to their high detection limits (approximately 105 CFU/mL for oCelloScope and 107 CFU/mL for Bioscreen C), the inability to distinguish between live and dead bacteria and early morphological changes of bacteria during exposure to ciprofloxacin, ertapenem and meropenem. Regrowth was more frequently detected in time-kill experiments, possibly related to the larger working volume with an increased risk of preexisting or emerging resistance. In comparison with Bioscreen C, the oCelloScope provided additional information on bacterial growth dynamics in the range of 105 to 107 CFU/mL and morphological features. In conclusion, the oCelloScope would be suitable for detection of in vitro effects of antibiotics, especially when a large number of regimens need to be tested.
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| 69. |
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| 70. |
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| 71. |
- Wang, Mei, et al.
(författare)
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T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages
- 2004
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 59:1, s. 53-69
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Tidskriftsartikel (refereegranskat)abstract
- The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes. (C) 2004 Elsevier B.V. All rights reserved.
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| 72. |
- 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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