Browsing by Subject "qPCR"

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  • Malkamäki, S.; Näreaho, A.; Lavikainen, A.; Oksanen, A.; Sukura, A. (2019)
    Berries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set of primers based on the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene was designed and evaluated. To assess the efficacy of the assay, we spiked bilberries (Vaccinium myrtillus) with a known amount of E. multilocularis eggs. The detection limit for the assay using the NAD1_88 primer set was 4.37 × 10−5 ng/μl of E. multilocularis DNA. Under artificial contamination of berries, 50 E. multilocularis eggs were reliably detected in 250 g of bilberries. Analytical sensitivity of the assay was determined to be 100% with three eggs. As an application of the assay, 21 bilberry samples from Finnish market places and 21 bilberry samples from Estonia were examined. Previously described sieving and DNA extraction methods were used, and the samples were analyzed for E. multilocularis and E. canadensis DNA using semi-quantitative real-time PCR and a melting curve analysis of the amplified products. Echinococcus DNA was not detected in any of the commercial berry samples. This easy and fast method can be used for an efficient detection of E. multilocularis and E. canadensis in bilberries or other berries, and it is applicable also for fruits and vegetables. © 2019 The Authors
  • Azinheiro, Sarah; Kant, Krishna; Shahbazi, Mohammad-Ali; Garrido-Maestu, Alejandro; Prado, Marta; Dieguez, Lorena (2020)
    Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ∼10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.
  • Rinta-Kanto, J. M.; Pehkonen, K.; Sinkko, H.; Tamminen, M. V.; Timonen, S. (2018)
    In this study, the abundance and composition of prokaryotic communities associated with the inner tissue of fruiting bodies of Suillus bovinus, Boletus pinophilus, Cantharellus cibarius, Agaricus arvensis, Lycoperdon perlatum, and Piptoporus betulinus were analyzed using culture-independent methods. Our findings indicate that archaea and bacteria colonize the internal tissues of all investigated specimens and that archaea are prominent members of the prokaryotic community. The ratio of archaeal 16S rRNA gene copy numbers to those of bacteria was >1 in the fruiting bodies of four out of six fungal species included in the study. The largest proportion of archaeal 16S rRNA gene sequences belonged to thaumarchaeotal classes Terrestrial group, Miscellaneous Crenar-chaeotic Group (MCG), and Thermoplasmata. Bacterial communities showed characteristic compositions in each fungal species. Bacterial classes Gammaproteobacteria, Actinobacteria, Bacilli, and Clostridia were prominent among communities in fruiting body tissues. Bacterial populations in each fungal species had different characteristics. The results of this study imply that fruiting body tissues are an important habitat for abundant and diverse populations of archaea and bacteria.
  • Malkamäki, Sanna; Näreaho, Anu Susanna; Oksanen, Antti; Sukura, Antti Kalle Kalervo (2019)
    Potential role of wild forest berries as a transmission vehicle for taeniid eggs was examined using non-zoonotic Taenia laticollis eggs as a model. The berries studied were bilberries (Vaccinium myrtillus) (1 m2 plot, n = 10) and lingonberries (Vaccinium vitis-idaea) (1 m2 plot, n = 11). The plots in the managed forest were evenly sprayed with 30,000 or 60,000 T. laticollis eggs suspended in water, and berries were collected 24 h after spraying. The berries were rinsed with water, and the water was sieved through a 1-mm and a 63-μm sieve to remove coarse material and through a 20-μm sieve to collect possible eggs. A small proportion of the sieved material was examined by microscopy after treatment with fluorescent Calcofluor White stain, which binds to eggshell chitin. In the recovery tests in artificially spiked samples, the detection limit was 5 eggs in 100 g of commercial frozen bilberries and lingonberries. Taeniid eggs were detected in all of the 10 experimentally contaminated bilberry samples and in 10 of 11 lingonberry samples. The sieved debris was also analyzed for T. laticollis DNA using semi-quantitative PCR. All samples were positive in quantitative SYBR Green real-time PCR using a T. laticollis-specific primer pair amplifying a short fragment of mitochondrial NADH dehydrogenase subunit 1 gene. This indicates that forest berries contaminated in shrubs contained T. laticollis eggs, and that berries can serve as a vehicle for taeniid eggs and may pose a possible risk to humans.
  • Pussila, Susanna (Helsingin yliopisto, 2014)
    The development of new antibiotics is very challenging work. However, it has become less attractive area of research for the pharmaceutical industry due to the rapid development of antibiotic resistance among bacteria. As a result, nowadays there are less new antimicrobial medicines appearing on the market. Increase in antibiotic resistance challenges the effective treatment of infectious diseases. Therefore, it is important to know about the existence and prevalence of antibiotic resistant bacteria and resistance genes. Waste water has been found to contain substantial amounts of antibiotics or their derivatives. Furthermore the bacterial density of the waste water is high. These factors may cause the selection pressure that assists the evolution, preservation and spread of antibiotic resistance in bacterial population. Wastewater treatment plants are believed to act as a reservoir of antibiotic resistant bacteria and antibiotic resistance genes. In this study, from samples taken from Viikinmäki wastewater treatment plant it was determined the quantity of resistance genes making bacteria resistance to a wide range of beta-lactam antibiotics. The samples were taken at the different stages of wastewater treatment process during three sampling days in June, September and December 2010. The samples were taken from the incoming wastewater and, from the final effluent and from the dried sludge. The DNA from wastewaters and sludge was isolated and the gene copy numbers of blaCTX-M-32 and blaOXA-58 antibiotic resistance genes were measured by quantitative PCR method. Antibiotic resistance gene copy numbers were normalized with 16S ribosomal RNA gene copy numbers. The antibiotic resistance genes investigated in this work make bacteria resistant to broad-spectrum penicillins, different groups of cephalosporins, monobactams, and carbapenems. It was found that blaOXA-58 and blaCTX-M-32 gene copies do exist in both incoming wastewater and dried sludge. From the incoming wastewater we found on average 3x10-3 blaOXA-58 gene copies/16S rRNA gene and 7x10-5 blaCTX-M-32 gene copies/16S rRNA gene. From dried sludge we found on average 3x10-3- blaOXA-58 gene copies/16S rRNA gene and 2x10-2 blaCTX-M-32 gene copies/16S rRNA. Both antibiotic resistance genes were also found in the final effluents. The amounts of antibiotic resistance genes /16S rRNA genes were found to decrease during of the wastewater treatment. In the final effluent the amount of blaOXA-58 genes was found to be two orders of magnitude less than in the incoming wastewater and for, blaCTX-M-32 gene copies it was found the decrease by one order of magnitude. Although the amount of antibiotic resistance genes decreased during of the wastewater treatment, the amount left in the final effluent is still notable and when it ends up in the water or soil it might have an effect to the antibiotic resistance in the nature.
  • Hyytiäinen, Heidi K; Jayaprakash, Balamuralikrishna; Kirjavainen, Pirkka V; Saari, Sampo E; Holopainen, Rauno; Keskinen, Jorma; Hämeri, Kaarle; Hyvärinen, Anne; Boor, Brandon E; Täubel, Martin (BioMed Central, 2018)
    Abstract Background Floor dust is commonly used for microbial determinations in epidemiological studies to estimate early-life indoor microbial exposures. Resuspension of floor dust and its impact on infant microbial exposure is, however, little explored. The aim of our study was to investigate how floor dust resuspension induced by an infant’s crawling motion and an adult walking affects infant inhalation exposure to microbes. Results We conducted controlled chamber experiments with a simplified mechanical crawling infant robot and an adult volunteer walking over carpeted flooring. We applied bacterial 16S rRNA gene sequencing and quantitative PCR to monitor the infant breathing zone microbial content and compared that to the adult breathing zone and the carpet dust as the source. During crawling, fungal and bacterial levels were, on average, 8- to 21-fold higher in the infant breathing zone compared to measurements from the adult breathing zone. During walking experiments, the increase in microbial levels in the infant breathing zone was far less pronounced. The correlation in rank orders of microbial levels in the carpet dust and the corresponding infant breathing zone sample varied between different microbial groups but was mostly moderate. The relative abundance of bacterial taxa was characteristically distinct in carpet dust and infant and adult breathing zones during the infant crawling experiments. Bacterial diversity in carpet dust and the infant breathing zone did not correlate significantly. Conclusions The microbiota in the infant breathing zone differ in absolute quantitative and compositional terms from that of the adult breathing zone and of floor dust. Crawling induces resuspension of floor dust from carpeted flooring, creating a concentrated and localized cloud of microbial content around the infant. Thus, the microbial exposure of infants following dust resuspension is difficult to predict based on common house dust or bulk air measurements. Improved approaches for the assessment of infant microbial exposure, such as sampling at the infant breathing zone level, are needed.
  • Hyytiainen, Heidi K.; Jayaprakash, Balamuralikrishna; Kirjavainen, Pirkka V.; Saari, Sampo E.; Holopainen, Rauno; Keskinen, Jorma; Hämeri, Kaarle; Hyvarinen, Anne; Boor, Brandon E.; Taubel, Martin (2018)
    Background: Floor dust is commonly used for microbial determinations in epidemiological studies to estimate early-life indoor microbial exposures. Resuspension of floor dust and its impact on infant microbial exposure is, however, little explored. The aim of our study was to investigate how floor dust resuspension induced by an infant's crawling motion and an adult walking affects infant inhalation exposure to microbes. Results: We conducted controlled chamber experiments with a simplified mechanical crawling infant robot and an adult volunteer walking over carpeted flooring. We applied bacterial 16S rRNA gene sequencing and quantitative PCR to monitor the infant breathing zone microbial content and compared that to the adult breathing zone and the carpet dust as the source. During crawling, fungal and bacterial levels were, on average, 8- to 21-fold higher in the infant breathing zone compared to measurements from the adult breathing zone. During walking experiments, the increase in microbial levels in the infant breathing zone was far less pronounced. The correlation in rank orders of microbial levels in the carpet dust and the corresponding infant breathing zone sample varied between different microbial groups but was mostly moderate. The relative abundance of bacterial taxa was characteristically distinct in carpet dust and infant and adult breathing zones during the infant crawling experiments. Bacterial diversity in carpet dust and the infant breathing zone did not correlate significantly. Conclusions: The microbiota in the infant breathing zone differ in absolute quantitative and compositional terms from that of the adult breathing zone and of floor dust. Crawling induces resuspension of floor dust from carpeted flooring, creating a concentrated and localized cloud of microbial content around the infant. Thus, the microbial exposure of infants following dust resuspension is difficult to predict based on common house dust or bulk air measurements. Improved approaches for the assessment of infant microbial exposure, such as sampling at the infant breathing zone level, are needed.
  • Virtanen, Jenni; Aaltonen, Kirsi; Vapalahti, Olli; Sironen, Tarja (2020)
    Aleutian disease (AD), caused by Aleutian mink disease virus (AMDV), causes significant welfare problems to mink, and financial losses to the farmers. As there is no vaccine or treatment available, reliable diagnostics is important for disease control. Here, we set up a probe-based real-time PCR (NS1-probe-PCR) to detect all strains of AMDV. PCR was validated and compared to two other real-time PCR methods (pan-AMDV- and pan-AMDO-PCR) currently used for AMDV diagnostics in Finland. The NS1-probe-PCR had a similar detection limit of 20 copies/reaction based on plasmid dilution series, and similar or better diagnostic sensitivity, when evaluated using spleen samples from mink, and stool samples from mink and foxes. None of the three PCR tests cross-reacted with other parvoviruses. The NS1-probe-PCR also showed a significantly higher specificity than the pan-AMDO-PCR with spleen samples and the best specificity with stool samples. Furthermore, it produced the results more rapidly than the other two PCRs making it a promising tool for both diagnostic and research purposes.
  • Pulkkinen, Katja; Pekkala, Nina; Ashrafi, Roghaieh; Hamalainen, Dorrit M.; Nkembeng, Aloysius N.; Lipponen, Anssi; Hiltunen, Teppo; Valkonen, Janne K.; Taskinen, Jouni (2018)
    Understanding ecological and epidemiological factors driving pathogen evolution in contemporary time scales is a major challenge in modern health management. Pathogens that replicate outside the hosts are subject to selection imposed by ambient environmental conditions. Increased nutrient levels could increase pathogen virulence by pre-adapting for efficient use of resources upon contact to a nutrient rich host or by favouring transmission of fast-growing virulent strains. We measured changes in virulence and competition in Flavobacterium columnare, a bacterial pathogen of freshwater fish, under high and low nutrient levels. To test competition between strains in genotype mixtures, we developed a quantitative real-time PCR assay. We found that a virulent strain maintained its virulence and outcompeted less virulent strains independent of the nutrient level and resource renewal rate while a less virulent strain further lost virulence in chemostats under low nutrient level and over long-term serial culture under high nutrient level. Our results suggest that increased outside-host nutrient levels might maintain virulence in less virulent strains and increase their contribution to epidemics in aquaculture. The results highlight a need to further explore the role of resource in the outside-host environment in maintaining strain diversity and driving evolution of virulence among environmentally growing pathogens.
  • Jokinen, Maija (Helsingin yliopisto, 2019)
    Parvoviruses are among the smallest known viruses. The parvovirus genome is a single stranded DNA, approximately 5 kb in size. The virion has a small (20 to 30 nm), rugged, non-enveloped icosahedral capsid. Parvoviruses can cause a number of diseases. Possibly the most recognized human parvovirus is parvovirus B19 (B19V), which can cause the so-called fifth disease, anemias and fetal death. Another relatively well characterised parvovirus is human bocavirus 1 (HBoV1), which causes respiratory tract infections in young children. Bufavirus (BuV) tusavirus (TuV) and cutavirus (CuV) are emerging parvoviruses, discovered during the years 2012-2016 using next generation sequencing methods. All three viruses were originally discovered in feces of patients suffering from diarrhea. BuV was originally found in Burkina Faso and has since been detected in fecal samples with polymerase chain reaction (PCR)-based methods from Europe, Asia and Africa. The seroprevalence of BuV differs between countries. TuV was found in a single stool sample from Tunisia, but no further reports of it have since emerged. CuV was found in 2016 and it has been linked to cutaneous T-cell lymphoma, but it is not known if the virus is the cause of the cancer or if the virus simply prefers quickly dividing cancer cells for its replication. BuV, TuV and CuV belong to the Protoparvovirus genus, but it is still unclear whether TuV is a human pathogen. More research is needed to study the epidemiology of these viruses and their role in illnesses. There were two main aims in this thesis: to set up an IgM µ-capture enzyme immunoassay (EIA) for human protoparvoviruses using BuV1 as an example and to screen three stool sample cohorts for BuV, TuV and CuV using an in-house multiplex quantitative PCR (qPCR). The IgM EIAs developed for B19V and HBoV1 was used as the base for developing human protoparvovirus IgM EIA, using Virus-like particles (VLP) as antigens. Setting up the EIA required a great amount of optimization and finally troubleshooting, since the assay did not work as expected. The troubleshooting revealed that the ambiguous results in the IgM µ-capture EIA were possibly due to degraded VLPs or that the sensitive µ-capture format requires extremely carefully purified VLPs. More optimizing is needed for this assay, however, the work done in this thesis offers a good base for further development of protoparvovirus IgM EIA. All three viruses were found in the stool samples during multiplex qPCR screening. Based on the qPCR and sequencing results one sample was positive for BuV DNA, one sample for TuV DNA and a total of 12 samples for CuV DNA. This is the first time TuV DNA has been found since its discovery. In addition to that, CuV DNA was identified in fecal samples for the first time since the discovery, previously CuV DNA had been found mostly in skin biopsies. As for TuV, based on the parvovirus phylogenetic analyses, its sequence is more closely related to rodent parvoviruses than CuV or BuV. More research is needed, possibly with animal and human samples, to establish the role of TuV as a human virus.
  • Puntila-Dodd, R.; Bekkevold, D.; Behrens, J. W. (Springer, 2021)
    Hydrobiologia 848: 2
    Species invasions often occur on coasts and estuaries where abiotic conditions vary, e.g. salinity, temperature, runoff etc. Successful establishment and dispersal of non-indigenous species in many such systems are poorly understood, partially since the species tend to show genetic and ecological plasticity at population level towards many abiotic conditions, including salinity tolerance. Plasticity may be driven by shifting expression of heat shock proteins such as Hsp70, which is widely recognized as indicator of physical stress. In this study, we developed a qPCR assay for expression of the hsp70 gene in the invasive round goby (Neogobius melanostomus) and tested the expression response of fish collected from a brackish environment in the western Baltic Sea to three different salinities, 0, 10 and 30. hsp70 expression was highest in fresh water, indicating higher stress, and lower at brackish (ambient condition for the sampled population) and oceanic salinities, suggestive of low stress response to salinities above the population’s current distribution. The highest stress in fresh water was surprising since populations in fresh water exist, e.g. large European rivers and Laurentian Great Lakes. The results have implications to predictions for the species’ plasticity potential and possible range expansion of the species into other salinity regimes.
  • Verhagen, Irene; Laine, Veronika N.; Mateman, A. Christa; Pijl, Agata; de Wit, Ruben; van Lith, Bart; Kamphuis, Willem; Viitaniemi, Heidi M.; Williams, Tony D.; Caro, Samuel P.; Meddle, Simone L.; Gienapp, Phillip; van Oers, Kees; Visser, Marcel E. (2019)
    The timing of breeding is under selection in wild populations as a result of climate change, and understanding the underlying physiological processes mediating this timing provides insight into the potential rate of adaptation. Current knowledge on this variation in physiology is, however, mostly limited to males. We assessed whether individual differences in the timing of breeding in females are reflected in differences in candidate gene expression and, if so, whether these differences occur in the upstream (hypothalamus) or downstream (ovary and liver) parts of the neuroendocrine system. We used 72 female great tits from two generations of lines artificially selected for early and late egg laying, which were housed in climate-controlled aviaries and went through two breeding cycles within 1 year. In the first breeding season we obtained individual egg-laying dates, while in the second breeding season, using the same individuals, we sampled several tissues at three time points based on the timing of the first breeding attempt. For each tissue, mRNA expression levels were measured using qPCR for a set of candidate genes associated with the timing of reproduction and subsequently analysed for differences between generations, time points and individual timing of breeding. We found differences in gene expression between generations in all tissues, with the most pronounced differences in the hypothalamus. Differences between time points, and early- and late-laying females, were found exclusively in the ovary and liver. Altogether, we show that fine-tuning of the seasonal timing of breeding, and thereby the opportunity for adaptation in the neuroendocrine system, is regulated mostly downstream in the neuro-endocrine system.
  • Pyöriä, Lari; Jokinen, Maija; Toppinen, Mari; Salminen, Henri; Vuorinen, Tytti; Hukkanen, Veijo; Schmotz, Constanze; Elbasani, Endrit; Ojala, Päivi M.; Hedman, Klaus; Välimaa, Hannamari; Perdomo, Maria F. (2020)
    Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LOD95 of similar to 10 to similar to 17 copies/reaction), with a dynamic range of 10' to 10 6 copies/p.I. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs. IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.
  • Lääveri, Tinja; Pakkanen, Sari H.; Antikainen, Jenni; Riutta, Jukka; Mero, Sointu; Kirveskari, Juha; Kantele, Anu (2014)
  • Gawriyski, Lisa (Helsingin yliopisto, 2018)
    Life historyresearch seeks to explain how natural selection and ecological challenges shape organisms to optimize their fitness. A strong immune defense is energetically demanding to upkeep and there may be trade-offs among other life history traits. Investing a lot of energy to upkeep a strong immune defense in conditions where there are less pathogens and parasites might have negative fitness effects. Heliconius eratois a neotropical species of butterfly found widely in South America. The immune defense, ecologicalfactors affecting its immune defense, and possible life history trade-offs of the butterfly are currently not well known. Environmental moisture conditions have been shown to affect the diversity, quality and amount of microorganisms and parasites. The aim of this thesis was to use real-time quantitative PCR (RT-qPCR) to quantify immune gene expression of individuals of the butterfly species Heliconius eratocollected from different environmental moisture conditions. Additionally, individual variation in encapsulation rates, a physiological measure of immunity, was compared across the moisture gradient. Results indicate reduced expression of the gene encoding the antimicrobial peptide attacin in dry conditions, but no difference in encapsulation rates across the moisture gradient. Additionally, differential expression of the prophenoloxidase encoding gene was found between male and female butterflies. These results indicate a possibility of differential immune threats in different environmental moisture conditions in H. erato, but requires further study.
  • König, Walter; Konig, Emilia; Weiss, Kirsten; Tuomivirta, Tero T.; Fritze, Hannu; Elo, Kari; Vanhatalo, Aila; Jaakkola, Seija (2019)
    BACKGROUND Nitrite and hexamine are used as silage additives because of their adverse effects on Clostridia and Clostridia spores. The effect of sodium nitrite and sodium nitrite/hexamine mixtures on silage quality was investigated. A white lupin-wheat mixture was treated with sodium nitrite (NaHe0) (900 g t(-1) forage), or mixtures of sodium nitrite (900 g t(-1)) and hexamine. The application rate of hexamine was 300 g t(-1) (NaHe300) or 600 g t(-1) (NaHe600). Additional treatments were the untreated control (Con), and formic acid (FA) applied at a rate of 4 L t(-1) (1000 g kg(-1)). RESULTS Additives improved silage quality noticeably only by reducing silage ammonia content compared with the control. The addition of hexamine to a sodium nitrite solution did not improve silage quality compared with the solution containing sodium nitrite alone. The increasing addition of hexamine resulted in linearly rising pH values (P <0.001) and decreasing amounts of lactic acid (P <0.01). Sodium nitrite based additives were more effective than formic acid in preventing butyric acid formation. Additives did not restrict the growth of Saccharomyces cerevisiae compared to the control. CONCLUSION The addition of hexamine did not improve silage quality compared with a solution of sodium nitrite. (c) 2018 Society of Chemical Industry
  • Hyytiäinen, Tiina (Helsingfors universitet, 2011)
    The human gastrointestinal tract (GIT) contains a complex microbiota which starts to develop after birth. Various factors such as age, health, diet and medication affect the composition of the GIT microbiota. The number and types of bacteria are different in each part of the GIT, but most of the bacteria are anaerobic. In faeces the number of bacterial cells is as high as 1011-1012 cfu/ml. The normal intestinal microbiota is essential for intestinal development, protein and carbohydrate metabolisms, and protection against pathogens. Sulphate-reducing bacteria (SRB) are typically anaerobic bacteria which use sulphate as a terminal electron acceptor to produce sulphide in their metabolism. Sulphate-reducing bacteria are widespread in all ecosystems including fresh water and marine sediments but are also present in the GIT. Most of SRB species are Gram-negative and they can use more than hundred compounds as electron donors. Dissimilatory sulphite reduction (dsrAB) gene is essential in sulphate reduction. dsrAB-gene encodes the enzyme called dissimilatory sulphite reductase, which is a key enzyme in the reduction of sulphite to sulphide. Recent findings suggest that SRB may have a role in human diseases, e.g. in periodontitis and inflammatory bowel disease (IBD). Connection between these disorders and SRB may be due to the highly toxic hydrogen sulphide. The aim of this study was to develop PCR-DGGE and qPCR methods for monitoring of sulphate-reducing bacteria from human faecal microbiota. In this study we used dsrAB-gene specific primers, which were used successfully in previous environmental microbiology studies. Previously published dsrAB-specific primers were used for PCR-DGGE. However, besides positive controls, two negative controls also amplified regardless of the modifications on temperature, amplification times, primers and MgCl2 concentration. In qPCR, specific and sensitive amplification was attained by using dsrA-gene specific primers. When the samples from paediatric patients with IBD (Crohn’s disease and ulcerative colitis) and healthy children were amplified, no differences were found between different disease groups. However there was a statistically significant difference (P <0.05) between the paediatric patients with Crohn’s disease who were on remission and those patients who’s disease was active (number of SRB; active<remission).
  • Fu, Yu (Helsingin yliopisto, 2017)
    Parvoviruses are among the smallest known viruses with a genome of ~ 5 kilobases. To date, six parvoviruses have been identified in human samples, with parvovirus B19 and human bocavirus being the only two known human pathogens in this family. Bufavirus, tusavirus and cutavirus are the most recently discovered parvoviruses, all belonging to the Protoparvovirus genus. Bufavirus was predominately discovered in fecal samples of children with diarrhea in Africa, Asia, and Europe. Cutavirus was detected in fecal samples of children with diarrhea in South America and Africa but has also been found in skin biopsies of patients with cutaneous T cell carcinoma and malignant melanoma. Tusavirus was discovered in a stool specimen of one child with diarrhea in Tunisia. At the moment, there are too little data to determine the identity of tusavirus as a human virus. More data and evidence are required to assess the association of the three parvoviruses with human diseases. In this study, a multiplex real-time PCR method was established to facilitate the detection and quantification of the three human-associated protoparvoviruses for further epidemiology and pathobiology study. Differentiation of different viruses was achieved by using three uniquely labeled probes. The multiplex assay was able to detect ≤ 10 copies/μL of bufavirus, tusavirus, and cutavirus plasmid templates simultaneously, with an average efficiency from 100.54% to 103.76%. The assay was applied to assess the prevalence of the three viruses in skin tissues of 93 non-immunosuppressed individuals with contact dermatitis and 137 immunosuppressed transplant recipients. Bufavirus and tusavirus DNA was detected in neither of the cohorts, which might indicate the rarity of the two viruses in skin tissues. Cutavirus DNA was detected in four (2.92%) transplant recipients, but all samples from the non-immunosuppressed group were negative. Among the four cutavirus positive patients, one was diagnosed with squamous cell carcinoma. These findings further support previous discoveries of cutavirus DNA in skin tissues and serve as evidence for the identity of cutavirus as a human virus. However, its association with cancer remains to be further investigated.
  • Kivivirta, Kimmo (Helsingfors universitet, 2016)
    Elintarvikeväärennösten havaitsemiseen käytetään DNA-pohjaisia tunnistusmenetelmiä. DNA-viivakoodaus on tunnistusmenetelmä, jolla pystytään toteamaan laji tuntemattomasta näytteestä lyhyen DNA-jakson eli viivakoodin perusteella. Tämänhetkiset viivakoodit kasveilla sisältävät nukleotiditasolla vähän lajien välisiä eroja, mikä heikentää näytteiden erottelukykyä ja näytteen oikeaa tunnistamista. Viivakoodien erottelukykyä arvioitiin eri puolukoiden (Vaccinium) suvun lajien kesken. Arviointi tehtiin viivakoodeista matK, ycf1, rpoC1 ja ITS. Puolukoiden suvulle ei löydetty viivakoodia, joka kykenisi tunnistamaan täydellisesti näytteitä lajitasolla. Kloroplastiset viivakoodit matK ja rpoC1 onnistuivat parhaiten PCR:ssa, monistamisessa ja sekvensoinnissa. Genomisen viivakoodin, ITS:n monistamisessa ilmeni häiriötä näytteissä, joissa oli vähän DNA:ta. Avoin lukukehys ycf1 puuttuu puolukoiden (Vaccinium) suvulta, mikä esti viivakoodin käyttämistä. DNA-viivakoodeista matK kykeni tunnistamaan BLAST-tietokantavertailussa seitsemän 14:sta näytteestä ja BOLD-tietokantavertailussa yhdeksän näytettä 14:sta näytteestä. ITS kykeni tunnistamaan BLAST-tietokantavertailussa kahdeksan näytettä 14:sta näytteestä. ITS:lle ei ollut riittävästi vastaavuuksia BOLD-tietokannassa ja rpoC1:lle ei kummassakaan tietokannassa. Sekvenssilinjauksessa matK ja rpoC1 olivat lajien välillä hyvin samankaltaisia. ITS sisälsi lajien välillä enemmän variaatiota, mutta tietokannoista puuttui vastaavia sekvenssejä, mistä syystä positiivisia tunnistuksia saatiin heikosti. DNA-viivakoodauksessa kasveilla kahden viivakoodin käyttö on edelleen suositeltavaa lajitason tunnistuksen tarkentamiseksi. Reaaliaikaisella PCR:lla (qPCR) pystytään spesifisesti toteamaan lajin läsnäolo näytteessä havaitsemalla DNA-jaksoja, jotka ovat ainutlaatuisia kohdelajin perimässä. Spesifinen tunnistusmenetelmä pyrittiin luomaan mustikalle (Vaccinium myrtillus) sijoittamalla alukkeet ja koetin lokuksille dfr ja MADS-box. Mustikan spesifisen menetelmän kehittelyssä ei saavutettu täydellistä spesifisyyttä. Mustikan lisäksi myös lajit V. praestans, V. smallii ja V. ovalifolium tuottivat positiivisen signaalin dfr-geenille suunnitelluilla alukkeilla ja koettimella. Positiivista ekspressiota tuottaneet lajit sisälsivät oletettavasti samankaltaisen dfr-geenin, jolle mustikalle spesifiset alukkeet ja koetin sitoutuivat. Spesifinen menetelmä kykeni kuitenkin sulkemaan pois muiden kasvisukujen näytteet sekä suurimman osan puolukoiden suvun näytteistä. Menetelmää voidaan hienosäätää kun dfr-geenin sekvenssi saadaan selville positiivisen signaalin antaneista lajeista tai kun muita suuremman muuntelun alueita paljastuu sekvensoinnin tuloksena.
  • Mäkilouko, Miia (Helsingin yliopisto, 2019)
    Antibioottien ylenmääräinen käyttö ja uusien antibioottien puute ovat johtaneet antibiooteille vastustuskykyisten bakteerien aiheuttamien sairauksien yleistymiseen. Lisääntynyt antibioottiresistenssi on maailmanlaajuinen ongelma, joka uhkaa globaalia terveyttä ja ruoan turvallisuutta. Staphylococcus aureus ja Pseudomonas aeruginosa ovat sairaaloissa yleisiä infektioita aiheuttavia mikrobeja. Metisilliiniresistentti S. aureus eli MRSA pystyy aiheuttamaan infektioita lähes missä tahansa kudoksessa. P. aeruginosa on akvaattisissa ympäristöissä yleinen mikrobi, joka on usein luonnollisesti vastustuskykyinen useille antibiooteille. Lisäksi molemmat bakteerit kykenevät biofilmin muodostamiseen, joka heikentää entisestään antibioottien tehoa. Jätevesien puhdistamoilla on yleisesti havaittu esiintyvän S. aureus- ja P. aeruginosa -bakteereita, mutta suurin osa tutkimuksista on keskittynyt tulevan ja käsitellyn jäteveden mikrobimääriiin ja/tai aktiivilietteeseen. Jätevesillä on ehdotettu olevan merkittävä rooli antibioottiresistenssin kehittymisessä ja leviämisesessä. Jätevesien puhdistamot keräävät yhteen kotitalouksien, teollisuuden ja sairaaloiden jätevesiä ja luovat niiden mukana tulleille mikrobeille tilaisuuden sekoittua ja vaihtaa geneettistä materiaalia, kuten antibioottiresistenssigeenejä. Toisaalta ne ovat myös paikkoja, joissa antibioottiresistenssejä bakteereita vastaan voi kehittyä uusia antimikrobiaalisia aineita tuottavia mikrobeja. Pro Gradu tutkielmani on osa TWIN-A konsortion hanketta ”uusia antibiootteja jätteistä”, jonka päämääränä on uusien antimikrobiaalisten aineiden löytäminen jätevesistä ja teollisista komposteista. Pro Gradu tutkielmassani kartoitan S. aureus ja P. aeruginosa bakteerien esiintymistä jätevedenpuhdistamoiden eri prosesseissa reaaliaika-PCR:n perusteella. Tutkimukseni tuloksia voidaan käyttää hankkeen jatkotutkimuksissa sekä jätevedenpuhdistamoiden riskinarvioinnissa. S. aureus -bakteeria kartoitettiin metisilliiniresistenttiä koodaavan mecA-geenin avulla ja S. aureus -bakteerille spesifistä nukleaasia koodaavan nucA-geenien avulla. P. aeruginosa -bakteeria kartoitettiin gyrB- ja ecfX-geenien avulla. Lisäksi näiden geenien kartoituksessa oli apuna koettimet.GyrB- ja ecfX-geeneissä olevilla muutoksilla on havaittu olevan vaikutusta bakteerin virulenssikykyyn. Kartoitettuja geenejä havaittiin esiintyvän yleisesti jätevedenpuhdistamojen prosesseissa, mutta pitoisuudet olivat alle määritysrajan. MecA-geenin esiintymisfrekvenssi oli nucA-geeniä suurempi, joka voi johtua siitä, että mecA-geeniä esiintyy myös muilla stafylokokki-lajeilla, kun nucA-geeni on spesifinen S. aureus-lajille. Myös gyrB-geenin esiintymisfrekvenssi oli korkeampi kuin ecfX-geenin, joka selittynee gyrB-geenin heikommalla lajispesifisyydellä. Kaikkien kartoitettujen geenien esiintyminen oli painottunut välppeeseen, aktiivilietteisiin, raakalietteeseen, palautuslietteeseen ja tiivistämölietteisiin. Välppeen läheinen kontakti ihmisen kanssa ja suuri orgaanisen aineen määrä selittävät korkeita esiintymisfrekvenssejä tässä prosessissa. Mikrobeille otolliset olot ja mikrobien sorptio aktiivilietteeseen selittävät kartoitettujen bakteerien yleisyyden aktiivilietteissä ja sen jälkeisissä prosesseissa mädättämölle asti. Mädättämöllä anaerobinen mädätys johtaa kartoitettujen geenien vähenemiseen. Mädättämöliete käsitellään Suomessa pääosin kompostoimalla, jossa lämpötilan nousu tappaa suurimman osan patogeeneistä. Kartoitettujen geenien poistuminen jätevedenpuhdistusprosesseista aktiivilietteen mukana, selittää myös geenien matalamman esiintymisfrekvenssin käsitellyssä jätevedessä verrattuna tulevaan jäteveteen. Tulosten perusteella kartoitetut bakteerit ja niiden antibioottiresistenssigeenit eivät aiheuta riskiä ympäristölle. Vaikka havaitut pitoisuudet olivat alle määritysrajan on kuitenkin hyvä pitää mielessä, että ympäristötekijöistä riippuen antibioottiresistenssigeenit ja bakteerit voivat kertyä ympäristöön ja sopivissa olosuhteissa lisääntyä ekspotentiaalisesti. Multiresistenssien bakteerien on myös havaittu selviävän paremmin jätevedenpuhdistusprosesseista, jonka vuoksi tilannetta olisi hyvä seurata tulevaisuudessa.