Browsing by Subject "qPCR"
Now showing items 1-7 of 7
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(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.
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(2022)The use of recycled fibers in paper production has increased during recent years. Recycled fibers are a more sustainable alternative compared to virgin fibers made from wood. However, paper mills utilizing recycled fibers have more microbiological problems compared to mills using only virgin fibers. Especially, anaerobic bacteria are harmful for papermaking processes utilizing recycled fibers. Bacteria of the class Clostridia comprise a very diverse group and have many different metabolic properties. Bacteria of class Clostridia can ferment different substrates, for example cellulose and starch, crucial in paper mills utilizing recycled fibers. Fermentation does not only decrease material efficiency, but also the acids produced during fermentation deteriorate papermaking processes. Volatile fatty acids are odorous compounds causing bad odors in the mills and in the final products. Clostridia can also produce, for example, hydrogen which is an explosive gas endangering the safety of the mill employees. Quantitative PCR is a feasible detection method for microbes. Here, a qPCR method was developed for the detection of most abundant bacteria in the class Clostridia in the recycled fiber mills. The designed primers targeted the most harmful bacteria from the genera Clostridium, Ethanoligenens, Fonticella and Ruminococcus identified in the recycled fiber mills. Three primer sets were designed for the target bacterial group. Positive controls of each target bacterial genus was included and close relatives from class Bacilli were used as negative controls. The designed primer sets were compared in efficiency, specificity and performance with process samples collected from paper mills using recycled fibers. One of the primer sets was found the most potential for the qPCR detection method for the diverse target bacterial group. All positive controls were amplified with the designed qPCR assay, whereas the designed primers discriminated well each negative control in vitro. The applicability of the designed qPCR assay was yet confirmed with process samples collected from mills utilizing recycled pulp. Even though the efficiency of the designed primer set was not optimal, the designed assay was determined feasible for the detection of the target group in the recycled fiber mills usually high in bacterial density.
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Insights on mechanical transmission of turnip mosaic virus (TuMV) mutants in Nicotiana benthamiana (2022)Turnip mosaic virus (TuMV) is an economically important plant RNA virus causing huge damage to wide range of arable and vegetable crops. A study was conducted in Nicotiana benthamiana to know if a TuMV mutant carrying a mutation in a thoroughly conserved WD-domain interacting motif and WG motif in HCPro protein can be mechanically transmitted to a healthy plant or not. HCPRoWD is a mutation in “AELPR” motif where glutamic acid and arginine are replaced by alanine. This mutated virus is here referred as TuMVWD. Similarly, in TuMVAG the tryptophan residue in the WG pair is changed to alanine and this mutated HCPro is called as TuMVAG. Four treatments, TuMVWT (positive control), Mock (negative control), TuMVWD and TuMVAG were made. Three plants per treatment were agroinfiltrated and five plants per treatment were used for mechanical inoculation experiment. Green fluorescent protein (GFP), a quantitative reporter of gene expression, was measured followed by qPCR for quantification of vRNA (viral RNA) accumulation. In agroinfiltrated plants, newly emerged leaves showed strong fluorescence in TuMVWT and TuMVAG by 14 dpi (days post inoculation), but TuMVWD showed poor GFP as compared to TuMVWT. During mechanical inoculation experiment, none of the treatments developed GFP in systemic leaves by six dpi but by 14 dpi GFP accumulation in the upper leaves of TuMVWT and TuMVAG was increased. TuMVWD was not used for 2nd mechanical experiment as it did not cause systemic infection during 1st mechanical inoculation experiment even by 14 dpi. Results from vRNA accumulation showed that mechanical transmission of virus was reduced with TuMVAG and not possible with TuMVWD. However, mutations had negative effect on vRNA accumulation.
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(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).
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(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.
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(2014)Glomus intraradices and Bacillus amyloliquefaciens are two commercially used plant growth promoting micro-organisms. They associate with plant roots to facilitate host plants to absorb nutrients, induce resistance against pathogens and pests, and regulate growth through phytohormones. Growth conditions for plants on green roofs are often unfavorable. In order to test whether growth and development of green roof plants could be enhanced via improving the microbial interface, G. intraradices and B. amyloliquefaciens were inoculated on experimental plots on a green roof in the summer of 2012. The experimental plots were marked as R (inoculated with B. amyloliquefaciens from Rhizocell), M (inoculated with G. intraradices from MYC4000), and C (control). The green roof was made of sedum-herb-grass mats. The plants included e.g. stonecrops, bluegrasses, yellow rockets, white clover, mullein, pennycress, and moss. The survival and development of G. intraradices and B. amyloliquefaciens were studied respectively from Poa alpina roots and soils in the summers of 2012 and 2013. G. intraradices was not detected in P alpina roots according to root staining and microscopy. Probable reasons for the lacking of G. intraradices include high phosphorus content in the soils, high soil temperature, and low soil moisture. PCR and qPCR were used to detect Bacillus content in green roof soils. The abundance of B. amyloliquefaciens was related to soil water content and soil temperature. During the last two measurements in 2012, 4 weeks of high moisture content in the soil resulted in large increase of B. amyloliquefaciens content in both M and R groups, but then decreased substantially due to drought and heat in 2013. In 2013, Only R group increased from the third to the last measurement, indicating probable resistance of the B. amyloliquefaciens strain from Rhizocell additive. The synergistic effect of B. amyloliquefaciens and G. intraradices might be responsible for the thousand-fold increase of Bacillus content in M group in 2012.
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(2022)Single nucleotide polymorphism (SNP) is DNA variation of a single nucleotide. SNPs are the most common mutations and millions of single nucleotide differences distinguish humans genetically from each other. Different gene variants of a single nucleotide affect nutritional and pharmacological metabolism and gene test results can therefore guide the diet and medication. SNPs are tested either by sequencing, gene arrays or quantitative polymerase chain reaction (qPCR). In this thesis the SNPs were tested by using 132 different TaqMan probes and the SmartChip qPCR platform. 20 participants donated two buccal swab samples and one dried blood spot (DBS) card. Buccal swabs were extracted using two different methods. DNA was also sent to a reference laboratory to be analysed by gene arrays and two participants also sent their DNA samples to two direct-to-consumer (DTC) commercial gene test companies. Low concentration buccal swab samples (<3 ng/µl) produced mismatches whereas DBS samples had high genotyping accuracy even at low DNA concentration. For buccal swabs there was a correlation between the DNA concentration and qPCR call rate (p<0.0001) but not for DBS samples (p>0.05). The allele separation of one TaqMan assay was not sufficient between minor homozygote and heterozygote clusters. For other 131 TaqMan assays excluding the low DNA concentration swab samples, the reproducibility rate was 99.97 %. The reproducibility rate with the DTC companies was 98.36 %. The lower reproducibility rate emphasizes the importance of the manual review of the genotyping raw data, which is not possible with huge sequencing or microarray SNP datasets. SNPs are found on various genetic structures which leads to inconsistent genotyping data that is difficult to analyse with a single algorithm. The SmartChip system combined with the TaqMan assays is a highly reproducible SNP genotyping method when the DNA concentration of the samples is high and the results are manually reviewed.
Now showing items 1-7 of 7