| 1. |
- Sidstedt, Maja, et al.
(författare)
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Digital PCR inhibition mechanisms using standardized inhibitors representing soil and blood matrices
- 2016
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentrationis determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrices such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. Here, we present how certain inhibitors disturb dPCR quantification and suggest solutions to these problems. Furthermore, we use real-time PCR, dPCR and isothermal titration calorimetry as tools to elucidate the mechanisms underlying the PCR inhibition. The impact of impurities on dPCR quantification was studied using humic acid as a model inhibitor. We show that the inhibitor-tolerance differs greatly for three different DNA polymerases, illustrating the importance of choosing a DNA polymerase-buffer system that is compatible with the samples to be analysed. Various inhibitory-substances from blood were found to disturb the system in different ways. For example, hemoglobin was found to cause quenching of fluorescence and a dramatic decrease of the number of positive reactions, leading to an underestimation of DNA quantity. IgG caused an increased number of late-starters. The system was more susceptible to inhibition by IgG when single-stranded DNA was used as template, compared with double-stranded DNA. By understanding more about the mechanisms of PCR inhibitors it will be possible to design more optimal PCR chemistries, improving dPCR detection and quantification.
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| 2. |
- Sidstedt, Maja, et al.
(författare)
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Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR
- 2018
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 410:10, s. 2569-2583
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Tidskriftsartikel (refereegranskat)abstract
- Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. [Figure not available: see fulltext.]
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| 3. |
- Sidstedt, Maja, et al.
(författare)
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The impact of common PCR inhibitors on forensic MPS analysis
- 2019
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Ingår i: Forensic Science International: Genetics. - : Elsevier BV. - 1872-4973. ; 40, s. 182-191
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Tidskriftsartikel (refereegranskat)abstract
- Massively parallel sequencing holds great promise for new possibilities in the field of forensic genetics, enabling simultaneous analysis of multiple markers as well as offering enhanced short tandem repeat allele resolution. A challenge in forensic DNA analysis is that the samples often contain low amounts of DNA in a background that may interfere with downstream analysis. PCR inhibition mechanisms of some relevant molecules have been studied applying e.g. real-time PCR and digital PCR. However, a detailed understanding of the effects of inhibitory molecules on forensic MPS, including mechanisms and ways to relieve inhibition, is missing. In this study, the effects of two well-characterized PCR inhibitors, humic acid and hematin, have been studied using the ForenSeq DNA Signature Prep kit. Humic acid and hematin resulted in lowered read numbers as well as specific negative effects on certain markers. Quality control of libraries with Fragment analyzer showed that increasing amounts of inhibitors caused a lowered amplicon quantity and that the larger amplicons were more likely to drop out. Further, the inhibitor tolerance could be improved 5–10 times by addition of bovine serum albumin in the initial PCR. On the contrary to the samples with inhibitors, low-template samples resulted in lowered read numbers for all markers. This difference strengthened the conclusion that the inhibitors have a negative effect on the DNA polymerase activity in the initial PCR. Additionally, a common capillary gel electrophoresis-based STR kit was shown to handle at least 200 times more inhibitors than the ForenSeq DNA Signature Prep kit. This suggests that there is room for improvement of the PCR components to ensure analytical success for challenging samples, which is needed for a broad application of MPS for forensic STR analysis.
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| 4. |
- Borgmästars, Emmy, et al.
(författare)
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Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing
- 2017
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Ingår i: Food and Environmental Virology. - : Springer Science and Business Media LLC. - 1867-0334 .- 1867-0342. ; 9:4, s. 395-405
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with Cq shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.
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| 5. |
- Chan, Sandy, et al.
(författare)
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Bacterial release from pipe biofilm in a full-scale drinking water distribution system
- 2019
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Ingår i: npj Biofilms and Microbiomes. - : Springer Science and Business Media LLC. - 2055-5008. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- Safe drinking water is delivered to the consumer through kilometres of pipes. These pipes are lined with biofilm, which is thought to affect water quality by releasing bacteria into the drinking water. This study describes the number of cells released from this biofilm, their cellular characteristics, and their identity as they shaped a drinking water microbiome. Installation of ultrafiltration (UF) at full scale in Varberg, Sweden reduced the total cell count to 1.5 × 10 3 ± 0.5 × 10 3 cells mL −1 in water leaving the treatment plant. This removed a limitation of both flow cytometry and 16S rRNA amplicon sequencing, which have difficulties in resolving small changes against a high background cell count. Following installation, 58% of the bacteria in the distributed water originated from the pipe biofilm, in contrast to before, when 99.5% of the cells originated from the treatment plant, showing that UF shifts the origin of the drinking water microbiome. The number of bacteria released from the biofilm into the distributed water was 2.1 × 10 3 ± 1.3 × 10 3 cells mL −1 and the percentage of HNA (high nucleic acid) content bacteria and intact cells increased as it moved through the distribution system. DESeq2 analysis of 16S rRNA amplicon reads showed increases in 29 operational taxonomic units (OTUs), including genera identified as Sphingomonas, Nitrospira, Mycobacterium, and Hyphomicrobium. This study demonstrated that, due to the installation of UF, the bacteria entering a drinking water microbiome from a pipe biofilm could be both quantitated and described.
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| 6. |
- Chan, Sandy, et al.
(författare)
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Monitoring biofilm function in new and matured full-scale slow sand filters using flow cytometric histogram image comparison (CHIC)
- 2018
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Ingår i: Water Research. - : Elsevier BV. - 0043-1354. ; 138, s. 27-36
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Tidskriftsartikel (refereegranskat)abstract
- While slow sand filters (SSFs) have produced drinking water for more than a hundred years, understanding of their associated microbial communities is limited. In this study, bacteria in influent and effluent water from full-scale SSFs were explored using flow cytometry (FCM) with cytometric histogram image comparison (CHIC) analysis; and routine microbial counts for heterotrophs, total coliforms and Escherichia coli. To assess if FCM can monitor biofilm function, SSFs differing in age and sand composition were compared. FCM profiles from two established filters were indistinguishable. To examine biofilm in the deep sand bed, SSFs were monitored during a scraping event, when the top layer of sand and the schmutzdecke are removed to restore flow through the filter. The performance of an established SSF was stable: total organic carbon (TOC), pH, numbers of heterotrophs, coliforms, E. coli, and FCM bacterial profile were unaffected by scraping. However, the performance of two newly-built SSFs containing new and mixed sand was compromised: breakthrough of both microbial indicators and TOC occurred following scraping. The compromised performance of the new SSFs was reflected in distinct effluent bacterial communities; and, the presence of microbial indicators correlated to influent bacterial communities. This demonstrated that FCM can monitor SSF performance. Removal of the top layer of sand did not alter the effluent water from the established SSF, but did affect that of the SSFs containing new sand. This suggests that the impact of the surface biofilm on effluent water is greater when the deep sand bed biofilm is not established.
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| 7. |
- Hedman, Johannes, et al.
(författare)
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Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics
- 2018
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Ingår i: Accreditation and Quality Assurance. - : Springer Science and Business Media LLC. - 0949-1775 .- 1432-0517. ; 23:3, s. 133-144
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Tidskriftsartikel (refereegranskat)abstract
- The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods.
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| 8. |
- Luhrig, Katharina, et al.
(författare)
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Bacterial Community Analysis of Drinking Water Biofilms in Southern Sweden
- 2015
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Ingår i: Microbes and Environments. - 1342-6311. ; 30:1, s. 99-107
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Tidskriftsartikel (refereegranskat)abstract
- Next-generation sequencing of the V1-V2 and V3 variable regions of the 16S rRNA gene generated a total of 674,116 reads that described six distinct bacterial biofilm communities from both water meters and pipes. A high degree of reproducibility was demonstrated for the experimental and analytical work-flow by analyzing the communities present in parallel water meters, the rare occurrence of biological replicates within a working drinking water distribution system. The communities observed in water meters from households that did not complain about their drinking water were defined by sequences representing Proteobacteria (82-87%), with 22-40% of all sequences being classified as Sphingomonadaceae. However, a water meter biofilm community from a household with consumer reports of red water and flowing water containing elevated levels of iron and manganese had fewer sequences representing Proteobacteria (44%); only 0.6% of all sequences were classified as Sphingomonadaceae; and, in contrast to the other water meter communities, markedly more sequences represented Nitrospira and Pedomicrobium. The biofilm communities in pipes were distinct from those in water meters, and contained sequences that were identified as Mycobacterium, Nocardia, Desulfovibrio, and Sulfuricurvum. The approach employed in the present study resolved the bacterial diversity present in these biofilm communities as well as the differences that occurred in biofilms within a single distribution system, and suggests that next-generation sequencing of 16S rRNA amplicons can show changes in bacterial biofilm communities associated with different water qualities.
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| 9. |
- Paul, Catherine J., et al.
(författare)
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Assessment of the mobile biofilm microbiome in distributed drinking water following installation of hybrid ultrafiltration process, in Varberg, Sweden.
- 2018
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Konferensbidrag (refereegranskat)abstract
- Drinking water is delivered from the treatment plant to the consumer through kilometres of pipes. Lining these pipes are communities of bacteria living as biofilms. Little is known about the microbial ecology of these biofilms, as access to drinking water pipes for sampling is often limited to pipes sporadically removed for repair or replacement. Bacteria are constantly exchanged between the biofilm and flowing water, however with bacteria in the water leaving the treatment plant reaching numbers as high as 700 000 cells per millilitre, identifying those cells originating from the biofilm is difficult.Membrane hybrid processes—coagulation coupled with ultrafiltration (UF)—have become a common method to comply with the legal, chemical, and microbiological requirements for drinking water. The main advantages of integrating coagulation with membrane filtration are the enhanced removal of natural organic matter (NOM) and reduced membrane fouling. Therefore, in November 2016, the Kvarnagården WTP in Varberg, Sweden was upgraded with a UF facility (capacity of 1080 m3 h−1 net permeate flow rate). The commissioning of the UF treatment process at Kvarnagården WTP, provided the opportunity to observe the microbial consequences of exposing a distribution system previously exposed to high cellular counts, to a virtually cell-free water phase. By observing which bacteria entered this new water phase, particularly those appearing after an extended time within the distribution system, a snapshot would be obtained of which bacteria can enter the water phase from the pipe biofilm.Water samples were taken before, three days after, and for one month after, installation of UF for flow cytometry (FCM) and Illumina 16S rRNA gene sequencing. FCM gives total cell counts and some additional parameters (%intact cells, %HNA) to describe the bacterial community, while DNA sequencing provides detailed genetic descriptions. Samples from the UF feed showed an average of 8 x105 cells/mL while filtered water contained 2.4 x 104 cells/mL. FCM also showed that UF removed intact cells, but that filtration by this method did not show a preference for either high (HNA) or low (LNA) nucleic acid-containing bacteria. Water samples from three locations at increasing distance from the treatment plant were taken within the distribution system. These samples showed an increase in total cells, with, on average, an addition of 0.6 x 104 cells/ mL, contributed from bacteria leaving the biofilm. These bacteria from the biofilm were further characterized by FCM as intact cells containing an increased percentage of HNA cells, relative to the water leaving the treatment plant. DNA sequencing of all samples, followed by the bioinformatics pipeline QIIME, further revealed the community present in the filtered water originating from the biofilm. Community content in the distributed water was distinct from the finished water, and also differed with respect to location within the distribution system. Both species richness and diversity in the distributed water decreased following installation of UF, as measured by Chao´s richness and Shannon diversity index. Differential analysis of the sequencing reads counts by DESeq2 were used to detect statistical significant operational taxonomic units (OTUs) that have changed over the travel across the pipes. Several groups of bacteria were identified associated with filtered water that had, and had not been, in contact with biofilm. These included Hyphomicrobium, Pedomicrobium, Nitrospira, Sphingomonas, Mycobacterium and Rhodobacter as well as several unidentified genera. This suggests that these are the most mobile members of the pipe biofilms in this distribution system. How the mobility of these groups change over seasons or how the overall microbiome of both the biofilm and the water phase in this distribution system will adapt to the filtered water over a longer time period will provide additional information about how pipe biofilms respond to large changes in the microbiology of distributed water.
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| 10. |
- Paul, Catherine J., et al.
(författare)
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Disturbance of the bacterial communities in drinking water produced from established and newly-built slow sand filters
- 2016
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Konferensbidrag (refereegranskat)abstract
- IntroductionSlow sand filtration is an established method for drinking water treatment, however, the knowledge about their microbial ecology remains limited. Maintenance of slow sand filters (SSF) includes washing to remove biofilm growth in the sand and restore flow through the filter. Understanding how this impacts produced water is important as the SSF may influence the bacterial flora in the finished drinking water and distribution system biofilms (El-Chakhtoura et al. 2015, Lührig et al. 2016, manuscript in preparation). Routine SSF monitoring after washing includes heterotrophic plate count (HPC) however this only describes a small fraction of the bacteria in the water (Allen, Edberg et al. 2004). Flow cytometry (FC) has been proposed for routine monitoring of bacteria in drinking water. FC counts the cells present in water and generates a fingerprint based on describing differential DNA staining to describe the population of bacteria (Arnoldini et al. 2013). Two full-scale new SSFs were constructed using different types of sand as starting material. Traditional microbial parameters (HPC, coliforms and E. coli) and FC data suggested these SSFs differed from each other and were not performing as well as established SSFs (Chan et al. 2016, manuscript in preparation). This study explored: using FC for routine monitoring of SSFs during washing; and, if the response to washing differed between each of the newly built SSFs when compared to an established SSF. Materials and MethodsInfluent and effluent water were characterized using HPC, the Colilert method (E.coli and coliforms), and FC according to established protocols (Prest et al. 2013). FC data was analyzed with Cytometric Histogram Image Comparison (CHIC) (Koch et al. 2013). Results and ConclusionsTwo new SSFs were built using only new sand (SSF-new), or a mix of new sand with sand previously removed during washing from existing sand filters (SSF-mix). After 6 months, the impact on water quality of disturbance from SSF washing was determined by comparing the bacterial populations of the influent and effluent water from SSF-new, SSF-mix, and an established SSF (SSF-est). No significant log reduction of cells was observed in any effluent water. The fluorescence distribution of effluent differed from that of the influent with each SSF showing a unique fingerprint (Fig 1.1). Figure 1.1 Comparison of fluorescence fingerprints for (left to right) SSF-est, SSF-mix, and SSF-new. Profiles show the combined plot of cell concentration and fluorescence distribution of nucleic acid stained with SYBR Green I in effluent water before wash (blue), and 4 days after washing (red).Fingerprints of effluent water were stable throughout the sampling period for SSF-est, regardless of washing, while for SSF-new and SSF-mix, fingerprints suggested a destabilization of the bacterial population related to washing. This suggested that SSF-new, SSF-mix and SSF-est differed in the population of cells being seeded into their respective effluent waters and this was supported by results from traditional methods and qPCR, showing lowest number of coliforms in the effluent from SSF-est. Analysis by CHIC, showed that the cell populations seeded by SSF-mix were more similar to those from SSF-est, than SSF-new. FC fingerprints from SSF-new were very similar to those of the influent water, suggesting that the biofilm in this SSF had little impact on the water passing through SSF-new. Since SSF-mix included washed sand from established SSFs, this appears to have inoculated the SSF-mix biofilm in a way that was not observed for SSF-new. The fingerprints for SSF-est effluent remained stable, consistent with a biofilm that is resilient to disturbance, and coliform counts demonstrating uninterrupted SSF function. This study thus suggests that washed sand is preferable to new sand for SSF construction and that more than 6 months is required for a resilient SSF biofilm. While total cell numbers did not change significantly across any SSF, SSF-est selectively reduced HPC and coliforms suggesting that this biofilm function is selective compared to newer SSF biofilms. In this study FC was not only useful for monitoring total cell counts to follow changes during SSF washing, but showed a consistent effluent water fingerprint associated with a resilient and functional SSF biofilm. As this was not observed for the newly built SSFs, FC can follow both the character and establishment of SSF biofilms and should be helpful for analyzing the impacts of any potential events on SSF biofilm integrity.ReferencesAllen, M.J., Edberg, S.C. and Reasoner, D.J., (2004). Heterotrophic plate count bacteria—what is their significance in drinking water? International journal of food microbiology, 92(3), pp.265-274.Arnoldini, M., Heck, T., Blanco-Fernández, A. and Hammes, F., (2013). Monitoring of dynamic microbiological processes using real-time flow cytometry. PloS one, 8(11), p.e80117.El-Chakhtoura, J., Prest, E., Saikaly, P., van Loosdrecht, M., Hammes, F. and Vrouwenvelder, H., 2015. Dynamics of bacterial communities before and after distribution in a full-scale drinking water network. Water Research, 74, pp.180-190.Koch, C., Fetzer, I., Harms, H. and Müller, S., (2013). CHIC—an automated approach for the detection of dynamic variations in complex microbial communities. Cytometry Part A, 83(6), pp.561-567.Prest, E.I., Hammes, F., Kötzsch, S., Van Loosdrecht, M.C.M. and Vrouwenvelder, J.S., (2013). Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method. Water research, 47(19), pp.7131-7142.
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| 11. |
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| 12. |
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| 13. |
- Sanga, Malin, et al.
(författare)
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A panel of PCR-inhibitory reference materials for quality evaluation of multiplex STR analysis kits
- 2015
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Ingår i: Forensic Science International. - : Elsevier. - 1875-1768 .- 1875-175X. ; 5, s. e317-319
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Tidskriftsartikel (refereegranskat)abstract
- PCR inhibition is a critical parameter in forensic DNA analysis. Substances interfering with amplification of short tandem repeats (STRs) may generate partial and/or ambiguous DNA profiles. We present a strategy for developing a broad panel of PCR-inhibitory reference materials (RMs), representing common casework samples. Our panel, including solutions prepared from for example cigarettes, chewing gum and soil, is a tool for in-house validation and lot testing of STR systems. PowerPlex ESX 16 Fast System tolerated high levels of some RMs, but several substances caused amplification problems. Humic acid had a negative effect on amelogenin, moist snuff hindered amplification of longer fragments, and chewing gum caused generally lowered allele peak heights. Applying a broad panel of RMs ensures that a wide range of inhibitory substances are tested, giving an improved understanding of inhibitor tolerance and effects.
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| 14. |
- Schleich, Caroline, et al.
(författare)
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Mapping Dynamics of Bacterial Communities in a Full-Scale Drinking Water Distribution System Using Flow Cytometry
- 2019
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Ingår i: Water. - : MDPI AG. - 2073-4441. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Microbial monitoring of drinking water is required to guarantee high quality water and to mitigate health hazards. Flow cytometry (FCM) is a fast and robust method that determines bacterial concentrations in liquids. In this study, FCM was applied to monitor the dynamics of the bacterial communities over one year in a full-scale drinking water distribution system (DWDS), following implementation of ultrafiltration (UF) combined with coagulation at the drinking water treatment plant (DWTP). Correlations between the environmental conditions in the DWDS and microbial regrowth were observed, including increases in total cell counts with increasing retention time (correlation coefficient R = 0.89) and increasing water temperature (up to 5.24-fold increase in cell counts during summer). Temporal and spatial biofilm dynamics affecting the water within the DWDS were also observed, such as changes in the percentage of high nucleic acid bacteria with increasing retention time (correlation coefficient R = −0.79). FCM baselines were defined for specific areas in the DWDS to support future management strategies in this DWDS, including a gradual reduction of chloramine.
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| 15. |
- Sidstedt, Maja, et al.
(författare)
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Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:3, s. 1642-1649
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Tidskriftsartikel (refereegranskat)abstract
- Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to S U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.
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| 16. |
- Sidstedt, Maja, et al.
(författare)
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Humic substances cause fluorescence inhibition in real-time PCR.
- 2015
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 487, s. 30-37
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Tidskriftsartikel (refereegranskat)abstract
- Real-time PCR (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, i.e. amplification inhibition. Humic substances (HS) are well known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, i.e. quench the fluorescence signal of dsDNA binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I and SYTO 82, generating lowered amplification plots although amplicon production was unaffected. For EvaGreen, 500 ng HA quenched almost all fluorescence, whereas 1000 ng HA completely inhibited amplification when applying Immolase DNA polymerase with BSA. Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional, and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor, but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.
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| 17. |
- Sidstedt, Maja, et al.
(författare)
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In-house validation of MPS-based methods in a forensic laboratory
- 2019
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Ingår i: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768. ; 7:1, s. 635-636
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Tidskriftsartikel (refereegranskat)abstract
- Massively parallel sequencing (MPS) methods are increasingly applied in forensic casework. However, adequate validation guidelines are lacking. In this work, we describe our in-house validation of the ForenSeq DNA Signature Prep Kit (Verogen) for analysis of ancestry- and phenotype-informative SNPs. We also discuss in-house validation of MPS assays in general terms. When validating the SNP assay, we focused on the reliability of SNP genotype calls and the compatibility with commonly analysed sample types. Other issues, for example analytical thresholds and accuracy of the data prediction model were considered to be covered by the developmental validation of the kit. Our study included determination of (1) concordance, (2) limit of detection, (3) matrix effects, (4) repeatability, and (5) contamination risk. In conclusion, the MPS-based SNP assay showed overall adequate performance for single-source samples, with correct genotype calls. We welcome a broad discussion on how to perform in-house validation of MPS-based methods, as this is vital to ensure timely implementation of reliable assays in forensic laboratories.
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| 18. |
- Sihto, Henna Maria, et al.
(författare)
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Effect of sodium nitrite and regulatory mutations Δagr, ΔsarA, and ΔsigB on the mRNA and protein levels of staphylococcal enterotoxin D
- 2016
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Ingår i: Food Control. - : Elsevier BV. - 0956-7135. ; 65, s. 37-45
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Tidskriftsartikel (refereegranskat)abstract
- Staphylococcal food poisoning results from ingestion of enterotoxins produced by Staphylococcus aureus. Staphylococcal enterotoxin D (SED) is one of the most common toxins detected in S. aureus strains associated with intoxications. The effect of sodium nitrite on enterotoxin production has been only partly investigated, despite its wide usage in meat products. In addition, the factors influencing SED regulation are unclear. The aim of this study was to determine the effect of sodium nitrite on sed transcription and SED production, as well as the effect of regulatory mutations on SED protein levels. Temporal sed mRNA and SED protein levels were compared in LB and LB supplemented with 150 mg/L nitrite, and SED protein levels between wild type (wt) and isogenic regulatory mutants (Δ. agr, Δ. sarA, Δ. sigB) under control and sodium nitrite conditions. Relative sed mRNA levels of wt strains were higher in late stationary phase in the presence of nitrite compared to control conditions. However, SED protein levels were decreased in the presence of nitrite. In LB, Δ. agr mutants showed SED levels similar to the wt, while Δ. sarA mutants exhibited reduced and Δ. sigB mutants increased SED levels compared to the wt. In LB with sodium nitrite, SED levels of mutant strains were reduced similar to the wt strains, except for two Δ. agr mutants, in which SED levels were increased in the presence of nitrite. Overall, strain-specific variation with regard to the effect of regulatory mutations was observed. In addition, the data suggests that SED regulation may not be as tightly dependent on Agr as previously described.
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| 19. |
- Susilo, Yusak Budi, et al.
(författare)
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Reduced enterotoxin D formation on boiled ham in staphylococcus aureus Δagr mutant
- 2017
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Ingår i: Toxins. - : MDPI AG. - 2072-6651. ; 9:9
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Tidskriftsartikel (refereegranskat)abstract
- Staphylococcal food poisoning (SFP) is a common cause of foodborne illness worldwide, and enterotoxin D (SED) is one of the most frequent Staphylococcus aureus enterotoxins associated with it. It has been reported that the expression and formation of SED in S. aureus is regulated by the quorum sensing Agr system. In this study, the effect of agr deletion on sed expression in S. aureus grown on boiled ham was investigated. Growth, sed mRNA and SED protein levels in an S. aureus wild type strain and its isogenic Δagr mutant were monitored for 14 days at 22 °C. The results showed that although deletion of the agr gene did not affect the growth rate or maximum cell density of S. aureus on boiled ham, it had a pronounced effect on SED formation during the first 5 days of incubation. The SED concentration was not reflected in the amount of preceding sed transcripts, suggesting that sed transcription levels may not always reflect SED formation. The expression of RNAIII transcript, the regulatory signal of the Agr system, was also monitored. Similar transcription patterns were observed for RNAIII and sed. Surprisingly, in the Δagr mutant, sed expression was comparable to that in the wild type strain, and was thus unaffected by deletion of the Agr system. These results demonstrate that the Agr system appears to only partially affect SED formation, even in a real food environment.
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| 20. |
- Zeaki, Nikoleta, et al.
(författare)
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Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation
- 2015
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 3:3, s. 551-566
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Tidskriftsartikel (refereegranskat)abstract
- The prophage-encoded staphylococcal enterotoxin A (SEA) is recognized as the main cause of staphylococcal food poisoning (SFP), a common foodborne intoxication disease, caused by Staphylococcus aureus. Studies on the production of SEA suggest that activation of the SOS response and subsequent prophage induction affect the regulation of the sea gene and the SEA produced, increasing the risk for SFP. The present study aims to evaluate the effect of NaCl and sorbic acid, in concentrations relevant to food production, on SOS response activation, prophage induction and SEA production. The impact of stress was initially evaluated on steady state cells for a homogenous cell response. NaCl 2% was found to activate the SOS response, i.e., recA expression, and trigger prophage induction, in a similar way as the phage-inducer mitomycin C. In contrast, sorbic acid decreased the pH of the culture to a level where prophage induction was probably suppressed, even when combined with NaCl stress. The impact of previous physiological state of the bacteria was also addressed on cells pre-exposed to NaCl, and was found to potentially affect cell response upon exposure to further stress. The results obtained highlight the possible SFP-related risks arising from the use of preservatives during food processing.
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| 21. |
- Zeaki, Nikoleta, et al.
(författare)
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Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions.
- 2015
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Ingår i: Toxins. - : MDPI AG. - 2072-6651. ; 7:12, s. 5359-5376
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Tidskriftsartikel (refereegranskat)abstract
- The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards.
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