| 1. |
- Andersson, Ulrika, et al.
(author)
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Beta-glucose 1-phosphate-interconverting enzymes in maltose- and trehalose-fermenting lactic acid bacteria.
- 2002
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In: Environmental Microbiology. - : Wiley. - 1462-2920 .- 1462-2912. ; 4:2, s. 81-88
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Journal article (peer-reviewed)abstract
- Maltose and trehalose catabolic pathways are linked through their common enzyme, beta-phosphoglucomutase, and metabolite, beta-glucose 1-phosphate, in Lactococcus lactis. Maltose is degraded by the concerted action of maltose phosphorylase and beta-phosphoglucomutase, whereas trehalose is assimilated by a novel pathway, including the recently discovered enzyme, trehalose 6-phosphate phosphorylase, and beta-phosphoglucomutase. In the present study, 40 strains of lactic acid bacteria were investigated for utilization of metabolic reactions involving beta-glucose 1-phosphate. All genera of the low G+C content lactic acid bacteria belonging to the clostridial subbranch of Gram-positive bacteria were represented in the study. The strains, which fermented maltose or trehalose, were investigated for beta-phosphoglucomutase, maltose phosphorylase and trehalose 6-phosphate phosphorylase activity, as indications of maltose and trehalose catabolic pathways involving beta-glucose 1-phosphate interconversions. Eighty per cent of all strains fermented maltose and, of these strains, 63% were shown to use a maltose phosphorylase/beta- phosphoglucomutase pathway. One-third of the strains fermenting trehalose were found to harbour trehalose 6-phosphate phosphorylase activity, and these were also shown to possess beta-phosphoglucomutase activity. Mainly L. lactis and Enterococcus faecalis strains were found to harbour the novel trehalose 6-phosphate phosphorylase/beta-phosphoglucomutase pathway. As lower beta-glucose 1-phosphate interconverting enzyme activities were observed in the majority of glucose-cultivated lactic acid bacteria, glucose was suggested to repress the synthesis of these enzymes in most strains. Thus, metabolic reactions involving the beta-anomer of glucose 1-phosphate are frequently found in both maltose- and trehalose-utilizing lactic acid bacteria.
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| 2. |
- Andersson, Ulrika, et al.
(author)
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Physiological function of the maltose operon regulator, MalR, in Lactococcus lactis.
- 2002
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In: BMC Microbiology. - 1471-2180. ; 2
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Journal article (peer-reviewed)abstract
- BACKGROUND: Maltose metabolism is initiated by an ATP-dependent permease system in Lactococcus lactis. The subsequent degradation of intracellular maltose is performed by the concerted action of Pi-dependent maltose phosphorylase and beta-phosphoglucomutase. In some Gram-positive bacteria, maltose metabolism is regulated by a maltose operon regulator (MalR), belonging to the LacI-GalR family of transcriptional regulators. A gene presumed to encode MalR has been found directly downstream the maltose phosphorylase-encoding gene, malP in L. lactis. The purpose of this study was to investigate the physiological role of the MalR protein in maltose metabolism in L. lactis. RESULTS: A L. lactis ssp. lactis mutant, TMB5004, deficient in the putative MalR protein, was physiologically characterised. The mutant was not able to ferment maltose, while its capability to grow on glucose as well as trehalose was not affected. The activity of maltose phosphorylase and beta-phosphoglucomutase was not affected in the mutant. However, the specific maltose uptake rate in the wild type was, at its lowest, five times higher than in the mutant. This difference in maltose uptake increased as the maltose concentration in the assay was increased. CONCLUSION: According to amino acid sequence similarities, the presumed MalR is a member of the LacI-GalR family of transcriptional regulators. Due to the suggested activating effect on maltose transport and absence of effect on the activities of maltose phosphorylase and beta-phosphoglucomutase, MalR of L. lactis is considered rather as an activator than a repressor.
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| 3. |
- Blixt, Y., et al.
(author)
-
Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria
- 2003
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In: International Journal of Food Microbiology. - 0168-1605 .- 1879-3460. ; 83:1, s. 15-26
-
Journal article (peer-reviewed)abstract
- A random amplified polymorphic DNA (RAPD) protocol was developed for interlaboratory use to discriminate food-borne Yersinia enterocolitica O:3 from other serogroups of Y. enterocolitica and from Y. enterocolitica-like species. Factors that were studied regarding the RAPD performance were choice of primers and concentration of PCR reagents (template DNA, MgCl 2, primer and Taq DNA polymerase). A factorial design experiment was performed to identify the optimal concentrations of the PCR reagents. The experiment showed that the concentration of the PCR reagents tested significantly affected the number of distinct RAPD products. The RAPD protocol developed was evaluated regarding its discrimination ability using 70 different Yersinia strains. Cluster analysis of the RAPD patterns obtained revealed three main groups representing (i) Y. pseudotuberculosis, (ii) Y. enterocolitica and (iii) Y. kristensenii, Y. frederiksenii, Y. intermedia and Y. ruckeri. Within the Y. enterocolitica group, the European serovar (O:3) and the North American serovar (O:8) could be clearly separated from each other. All Y. enterocolitica O:3 strains were found in one cluster which could be further divided into two subclusters, representing the geographical origin of the isolates. Thus, one of the subclusters contained Y. enterocolitica O:3 strains originating from Sweden, Finland and Norway, while Danish and English O:3 strains were found in another subcluster together with O:9 and O:5,27 strains. The repeatability (intralaboratory) and reproducibility (interlaboratory) of the RAPD protocol were tested using 15 Yersinia strains representing different RAPD patterns. The intralaboratory and the interlaboratory studies gave similarity coefficients of the same magnitude (generally >70%) for the individual strains. In the present study, it was shown that interreproducible RAPD results could be achieved by appropriate optimisation of the RAPD protocol. Furthermore, the study reflects the heterogeneous genetic diversity of the Y. enterocolitica species. © 2002 Elsevier Science B.V. All rights reserved.
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| 4. |
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| 5. |
- Dahlenborg, Maria, et al.
(author)
-
Prevalence of Clostridium botulinum types B, E, and F in faecal samples from Swedish cattle.
- 2003
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In: International Journal of Food Microbiology. - 0168-1605 .- 1879-3460. ; 82:2, s. 105-110
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Journal article (peer-reviewed)abstract
- Faeces were collected from 60 cows at three slaughterhouses situated in southern and central Sweden. The faecal samples were collected during two sampling periods over the year, summer and winter. All samples were analysed for the presence of Clostridium botulinum spores, according to a combined selection and enrichment PCR procedure. One PCR assay was specific for part of the type B neurotoxin gene, while the other assay was specific for both type E and F neurotoxin genes. The prevalence of C. botulinum in Swedish cattle was established to be 73% for non-proteolytic type B and less than 5% for types E and F. Twenty-eight (64%) of the positive faecal samples had a spore load of less than 4 spores/g. Statistical analysis (ANOVA) showed that seasonal variation (summer and winter) had a significant effect on the prevalence of C. botulinum type B in cattle, whereas the effect of geographical location of rearing of the cattle (southern and central Sweden) was less significant.
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| 6. |
- Hoorfar, Jeffrey, et al.
(author)
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Diagnostic PCR: validation and sample preparation are two sides of the same coin
- 2004
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In: APMIS : acta pathologica, microbiologica, et immunologica Scandinavica. - : Wiley. - 1600-0463. ; 112:11-12, s. 808-814
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Research review (peer-reviewed)abstract
- Increased use of powerful PCR technology for the routine detection of pathogens has focused attention on the need for international validation and preparation of official non-commercial guidelines. Bacteria of epidemiological importance should be the prime focus, although a "validation infrastructure" once established could easily be adapted for PCR-based detection of viruses and parasites. The aim of standardization should be the widespread adoption of diagnostic PCR for routine pathogen testing. European experience provides the impetus for realization of this vision through preparation of quantitative reference DNA material and reagents, production of stringent protocols and tools for thermal cycler performance testing, uncomplicated sample preparation techniques, and extensive ring trials for assessment of the efficacy of selected matrix/pathogen detection protocols.
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| 7. |
- Knutsson, Rickard, et al.
(author)
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Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation
- 2002
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In: International Journal of Food Microbiology. - 0168-1605. ; 72:3, s. 185-201
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Journal article (peer-reviewed)abstract
- A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation <5%. When a background flora was present at concentrations greater than or equal to10(6) (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment, Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10(1) (CFU/ml) Y enterocolitica in the presence of at least an inoculation concentration of 10(3) (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR. (C) 2002 Elsevier Science B.V. All rights reserved.
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| 8. |
- Knutsson, Rickard, et al.
(author)
-
Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica.
- 2002
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In: International Journal of Food Microbiology. - 0168-1605 .- 1879-3460. ; 73:1, s. 35-46
-
Journal article (peer-reviewed)abstract
- Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.
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| 9. |
- Knutsson, Rickard, et al.
(author)
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Modeling of 5′ nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica
- 2002
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In: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:1, s. 52-60
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Journal article (peer-reviewed)abstract
- The performance of a 5′ nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (Cτ) and the fluorescence intensity by a normalized reporter value (ΔRn). The Cτ response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTth. A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH2O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the rTth mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 × 10-12 g/microwell for the rTth mixture and 2 × 10-12 g/microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30°C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture resulted in an earlier PCR detection during enrichment than use of the AmpliTaq Gold mixture. For accurate detection (Cτ ≤ 30) of S. enterica serovar Enteritidis inoculated in BPW, the rTth mixture required 8.4 h of enrichment, while the AmpliTaq Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5′ nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures.
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| 10. |
- Lahiri, SD, et al.
(author)
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Crystallization and preliminary X-ray diffraction studies of beta-phosphoglucomutase from Lactococcus lactus
- 2002
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In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 58, s. 324-326
-
Journal article (peer-reviewed)abstract
- beta-Phosphoglucomutase (beta-PGM), a 28 kDa monomer, catalyzes the reversible conversion of beta-D-glucose-1-phosphate to beta-D-glucose-6-phosphate in maltose metabolism in a variety of organisms. Sequence analysis of beta-PGM indicates that it is a member of the haloacid dehalogenase (HAD) enzyme superfamily, which evolved to cleave C-Cl, C-P and C-OP bonds in a variety of substrates. beta-PGM has been crystallized using the hanging-drop method. Diffraction-quality crystals of the native protein have been obtained from two conditions, both belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.67, b = 92.78, c = 111.60 and a = 53.21, b = 57.01, c = 76.11 Angstrom. To solve the phase problem, selenomethionine (SeMet) containing alpha-PGM crystals have been grown. The SeMet-containing crystals diffract to high resolution only when grown by microseeding with native crystals. A three-wavelength data set has been collected to 2.3 Angstrom on crystals of the SeMet-substituted beta-PGM. The structure solution is currently being attempted by the multi-wavelength anomalous diffraction (MAD) phasing method.
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| 11. |
- Levander, Fredrik, et al.
(author)
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Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus.
- 2002
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In: Applied and Environmental Microbiology. - 0099-2240. ; 68:2, s. 784-790
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Journal article (peer-reviewed)abstract
- It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.
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| 12. |
- Lübeck, P S, et al.
(author)
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Towards an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.
- 2003
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In: Applied and Environmental Microbiology. - 0099-2240. ; 69:9, s. 5664-5669
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Journal article (peer-reviewed)abstract
- As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.
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| 13. |
- Löfström, Charlotta, et al.
(author)
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Rapid and Specific Detection of Salmonella spp. in Animal Feed Samples by PCR after Culture Enrichment
- 2004
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 70:1, s. 69-75
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Journal article (peer-reviewed)abstract
- A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.
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| 14. |
- Lövenklev, Maria, et al.
(author)
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Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B
- 2004
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 70:5, s. 2928-2934
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Journal article (peer-reviewed)abstract
- The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO2 in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO2 was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO2 concentration; the cntB mRNA level was fivefold greater in a 70% CO2 atmosphere than in a 10% CO2 atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng·ml -1·unit of optical density-1 in the 10% CO 2 atmosphere and 126 ng·ml-1·unit of optical density-1 in the 70% CO2 atmosphere.
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| 15. |
- Lövenklev, Maria, et al.
(author)
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Relative neurotoxin gene expression in Clostridium botulinum type B, determined using quantitative reverse transcription-PCR
- 2004
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 70:5, s. 2919-2927
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Journal article (peer-reviewed)abstract
- A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum. The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA. The patterns and relative expression of cntB were different in the different strains. Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase. In the proteolytic strain C. botulinum ATCC 7949, the level of cntB mRNA was four- to fivefold higher than the corresponding levels in the other strains. This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay. When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number. Instead, the level of cntB mRNA remained the same. When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred. In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations. This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in the cntB mRNA level was observed in all growth phases.
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| 16. |
- Malorny, B, et al.
(author)
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Standardization of diagnostic PCR for the detection of foodborne pathogens.
- 2003
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In: International Journal of Food Microbiology. - 0168-1605. ; 83:1, s. 39-48
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Journal article (peer-reviewed)abstract
- In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection of pathogenic bacteria in foods. The present review focuses on the harmonization procedure and standardization criteria for detection of foodborne pathogens by PCR. The progress of standardization so far and future perspectives of diagnostic PCR are discussed.
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| 17. |
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| 18. |
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| 19. |
- Palmfeldt, Johan, et al.
(author)
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Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis.
- 2003
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In: Cryobiology. - 0011-2240. ; 47:1, s. 21-29
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Journal article (peer-reviewed)abstract
- The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6 ± 0.2%) and sucrose the highest (6.4 ± 1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 × 107 to 5 × 1010 CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15–25%, were obtained for initial cell concentrations between 1 × 109 and 1 × 1010 CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26 ± 6%.
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| 20. |
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| 21. |
- Rådström, Peter, et al.
(author)
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Pre-PCR processing : Strategies to generate PCR-compatible samples
- 2004
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In: Molecular Biotechnology. - 1073-6085 .- 1559-0305. ; 26:2, s. 133-146
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Journal article (peer-reviewed)abstract
- Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.
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| 22. |
- Rådström, Peter, et al.
(author)
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Pre-PCR processing of samples.
- 2003
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In: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 216
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Journal article (peer-reviewed)
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| 23. |
- Rådström, Peter, et al.
(author)
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Pre-PCR processing strategies
- 2004
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In: PCR technology : current innovations. - 0849311845
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Book chapter (other academic/artistic)abstract
- A review describes the sample prepn. for biochem. methods, enrichment methods, immunol. methods, and phys. methods. The pretreatment of a complex biol. sample is crucial, and for successful PCR the following requirements have to be fulfilled: complete lack or low concn. of PCR-inhibitory components in the sample and sufficient concn. of target DNA. The aim of the pre-PCR treatment is to convert a complex biol. sample contg. the target microorganisms into PCR-amplifiable samples.
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| 24. |
- Wolffs, Petra, et al.
(author)
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Impact of DNA polymerases and their buffer systems on quantitative real-time PCR
- 2004
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In: Journal of Clinical Microbiology. - 1098-660X. ; 42:1, s. 408-411
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Journal article (peer-reviewed)abstract
- An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.
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| 25. |
- Wolffs, Petra, et al.
(author)
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Rapid quantification of yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR
- 2004
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In: Journal of Clinical Microbiology. - 1098-660X. ; 42:3, s. 1042-1047
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Journal article (peer-reviewed)abstract
- The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 +/- 0.021 and 1.007 +/- 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 +/- 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y enterocolitica was possible down to a level of at least 4.2 x 10(3) CFU per ml of meat juice, even in the presence of 10(6) CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.
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