Browsing by Subject "RT-qPCR"

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  • Gregorova, Pavlina; Heinonen, Minna-Maria Kristiina; Sarin, Peter (2022)
  • Varga, Zsuzsanna; Lebeau, Annette; Bu, Hong; Hartmann, Arndt; Penault-Llorca, Frederique; Guerini-Rocco, Elena; Schraml, Peter; Symmans, Fraser; Stoehr, Robert; Teng, Xiaodong; Turzynski, Andreas; von Wasielewski, Reinhard; Guertler, Claudia; Laible, Mark; Schlombs, Kornelia; Joensuu, Heikki; Keller, Thomas; Sinn, Peter; Sahin, Ugur; Bartlett, John; Viale, Giuseppe (2017)
    Background: Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER2/ERBB2), estrogen receptor (ER/ESR1), progesterone receptor (PgR/PGR), and marker of proliferation Ki67 (MKI67) is indispensable for therapeutic decision making in early breast cancer. In this multicenter prospective study, we addressed the issue of inter- and intrasite reproducibility using the recently developed reverse transcription-quantitative real-time polymerase chain reaction-based MammaTyper (R) test. Methods: Ten international pathology institutions participated in this study and determined messenger RNA expression levels of ERBB2, ESR1, PGR, and MKI67 in both centrally and locally extracted RNA from formalin-fixed, paraffin-embedded breast cancer specimens with the MammaTyper (R) test. Samples were measured repeatedly on different days within the local laboratories, and reproducibility was assessed by means of variance component analysis, Fleiss' kappa statistics, and interclass correlation coefficients (ICCs). Results: Total variations in measurements of centrally and locally prepared RNA extracts were comparable; therefore, statistical analyses were performed on the complete dataset. Intersite reproducibility showed total SDs between 0.21 and 0.44 for the quantitative single-marker assessments, resulting in ICC values of 0.980-0.998, demonstrating excellent agreement of quantitative measurements. Also, the reproducibility of binary single-marker results (positive/negative), as well as the molecular subtype agreement, was almost perfect with kappa values ranging from 0.90 to 1.00. Conclusions: On the basis of these data, the MammaTyper (R) has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise and reproducible quantitative assessment of the established breast cancer biomarkers and molecular subtypes in a decentralized workup.
  • Nykänen, Sonja (Helsingin yliopisto, 2019)
    Colorectal cancer (CRC) kills more than half a million people a year worldwide. Usually the disease develops over several years via multiple steps which involve both genetic and epigenetic alterations. CRC is often diagnosed at late stage, when the cancer has already metastasized, and the prognosis is relatively poor. Several studies suggest that the first changes towards colorectal cancer occur and can be detected in histologically normal tissue before the appearance of any detectable lesion. The precancerous cells harbouring those changes may form a field of tissue, which is predisposed to malignant transformation. The study of pre-cancerous tissue might reveal the earliest changes in CRC development, which can be used as biomarkers for early detection and prevention of CRC. The aim of this thesis was to revise and investigate whether the aberrant expression of the six chromosomal segregation genes, Bub1, Mis18a, Pms2, Rad9a, Tpx2, and Mlh1, would signal carcinogenesis in mouse colon mucosa. Altogether fourteen mice, of which six had a proximal colon carcinoma, were selected for the study. The expression analysis was performed to histologically normal colon mucosa collected from the proximal and distal colon of each mice in order to investigate whether the possible pre-cancerous changes are found exclusively in the close proximity to the carcinoma. The expression was quantified with reverse transcription quantitative polymerase chain reaction (RTqPCR). No statistically significant gene expression differences were found between the carcinoma and control mice, indicating that the studied mice did not display cancer-preceding expression changes of the six studied genes in the carcinoma adjacent histologically normal colon mucosa. The results differed from the previously reported results, where the expressions of the six genes were found to be downregulated in the carcinoma adjacent mucosa. Here, the sample size was presumably not large enough to reveal statistically significant clustering of the expression patterns. However, Bub1 seemed to have a downregulated trend in the carcinoma adjacent mucosa, which supports the previously suggested role of Bub1 alterations in CRC initiation.
  • Kaloinen, Minttu (Helsingin yliopisto, 2020)
    Since the discovery of Marburgvirus (MARV) in 1967, filoviruses have been identified as the causative agent for several outbreaks of hemorrhagic fever in equatorial Africa. High morbidity and mortality rates characterize especially Ebola virus disease (EVD) when Zaire ebolavirus (EBOV) is implicated. In Dec 2013, on outbreak of unprecedented magnitude started in West Africa, demanding international attention, aid and measures to restrict the spread of the epidemic. In Europe, the Ebola MoDERN APPROCHES FOR BEDSIDE RAPID DIAGNOSTICS (Ebola MoDRAD) project was set up to promote fast and accurate diagnosis of EVD. As part of this project, in this study six RT-qPCR assays (primer and probe sets or commercial kits) were compared to discover the best suitable for future development of field-deployable diagnostic test to be used in epidemics. Out of these methods, four are specific to ZEBOV NP (nucleoprotein) gene: assays by Weidmann et al., Trombley et al., Huang et al. and Clonit quanty ZEBOV Fast, and two multiplex assays targeting the L (RNA-dependent RNA polymerase) gene of several ebolaviruses (and marburgviruses): RealStar® Filovirus Screen RT-PCR Kit 1.0 by Altona Diagnostics as well as the assay by Jääskeläinen et al., which has subsequently been modified to include several ebolaviruses and Marburg marburvirus. The sample panel included in vitro produced RNA controls, inactivated whole viruses and negative controls. The analytical sensitivity of the assays was determined by Probit analysis for limit of detection (LOD) and the intra-assay repeatibility by calculating the cycle threshold coefficient variation percentage. All methods showed good specificity for their targets, yet the performance of the Clonit and Altona test kits was dependent on sample matrix. The golden standard assay by Trombley et al. was very sensitive, but the intra-assay covariance was also high (up to 13%). The assay by Jääskeläinen et al. was chosen for future development for the best overall reliability and the ability to detect several filoviruses.
  • Hartikainen, Anna (Helsingin yliopisto, 2019)
    Nykyinen jälkiteollinen ihmiskunta tuottaa energiansa pääasiassa fossiilisilla polttoaineilla. Pariisin ilmastosopimuksen osapuolet sitoutuivat pitämään ilmaston lämpenemisen alle kahdessa celsiusasteessa verrattuna esiteolliseen aikaan ja muuttamaan energiantuotantotapojaan. Yhtenä ratkaisuna nähdään uusiutuvat energianlähteet, joita kuuluvat muun muassa biopolttoaineet. Bioetanoli on maailmanlaajuisesti käytetyin liikenteen biopolttoaine, jonka tuotanto on huomattavasti kasvanut 1980-luvulta lähtien. Tällä hetkellä bioetanolia tuotetaan etupäässä ruoantuotannon kanssa kilpailevista sokeri- ja tärkkelyspitoisista viljelykasveista, mutta niin kutsuttujen toisen sukupolven biopolttoaineiden tuotanto olisi ihmiskunnan ja maapallon kannalta kestävämmällä pohjalla. Toisen sukupolven bioetanolia valmistetaan kasvi- ja puupohjaisesta jätemateriaalista, kuten maa- ja metsätalouden sekä elintarviketeollisuuden jätteistä. Aikaisemmissa tutkimuksissa hyväksi lignoselluloosan ja puuperäisten jätteiden hajottajaksi ja monipuoliseksi entsyymien tuottajaksi tunnistettu, valkolahottajiin kuuluva kääpämäinen kantasieni rusorypykkä Phlebia radiata kykenee samanaikaiseen kiinteän kasvualustan hajotukseen ja etanolifermentaatioon. Tämän vuoksi rusorypykän etanolin tuottoa ja siihen liittyviä perusaineenvaihduntareittejä haluttiin tutkia tarkemmin. Sienen rihmastoa kasvatettiin jätelignoselluloosamateriaaleilla hapettomiksi muuttuvissa fermentaatio-olosuhteissa. Etanolin tuoton ja kasvualustan pilkkoutumisen lisäksi tutkittiin käänteistranskription kvantitatiivisella PCR-reaktiolla lignoselluloosan hajotukseen ja etanolifermentaatioon liittyviä entsyymejä koodaavia geenejä ja näiden geenien ilmentymistä eri kasvuaikapisteissä. Tulosten perusteella havaittiin rusorypykän tuottavan noin 13,5 massa-% etanolia kasvualustan hiilihydraattien sokeripitoisuuden perusteella lasketusta teoreettisesta maksimiarvosta. Lignoselluloosan hajotukseen ja etanolin fermentointiin oleellisesti liittyvien entsyymien geenit ilmentyivät kasvatusten aikana jätelignoselluloosalla tilastollisesti merkitsevin eroin eri aikapisteissä, mikä osoitti hajotustoiminnan, rihmaston kasvun ja etanolin tuoton olevan muuttuvia ja toisiinsa vaikuttavia prosesseja. Sieni pystyi hyödyntämään kiinteää kasvualustaansa irrottaen siitä käyttöönsä sokereita. Rihmaston kasvusta ja elävästä sienibiomassasta viitteitä antavaa ergosterolia havaittiin koko kuukauden pituisen kasvatuksen ajan. Lisäksi kaasukehässä havaittiin odotettuja fermentaatiomuutoksia, kuten hiilidioksidin kertymistä, ja happikaasu oli neljässä viikossa lähes kulutettu loppuun. Tulosten perusteella rusorypykkä osoittautui tehokkaaksi jätelignoselluloosan pilkkojaksi ja kestäväksi etanolin tuottajaksi. RNA-eristys kiinteältä kasvualustalta sekä aineenvaihduntageenien ilmentymisen analysointi RT-qPCR-menetelmällä mahdollistivat rusorypykän transkriptomin jatkotutkimuksen geenien säätelyn selvittämiseksi fermentoivissa ja lähes hapettomissa olosuhteissa.
  • Wirtz, Ralph M.; Sihto, Harri; Isola, Jorma; Heikkila, Paivi; Kellokumpu-Lehtinen, Pirkko-Liisa; Auvinen, Paivi; Turpeenniemi-Hujanen, Taina; Jyrkkio, Sirkku; Lakis, Sotiris; Schlombs, Kornelia; Laible, Mark; Weber, Stefan; Eidt, Sebastian; Sahin, Ugur; Joensuu, Heikki (2016)
    The biological subtype of breast cancer influences the selection of systemic therapy. Distinction between luminal A and B cancers depends on consistent assessment of Ki-67, but substantial intra-observer and inter-observer variability exists when immunohistochemistry (IHC) is used. We compared RT-qPCR with IHC in the assessment of Ki-67 and other standard factors used in breast cancer subtyping. RNA was extracted from archival breast tumour tissue of 769 women randomly assigned to the FinHer trial. Cancer ESR1, PGR, ERBB2 and MKI67 mRNA content was quantitated with an RT-qPCR assay. Local pathologists assessed ER, PgR and Ki-67 expression using IHC. HER2 amplification was identified with chromogenic in situ hybridization (CISH) centrally. The results were correlated with distant disease-free survival (DDFS) and overall survival (OS). qPCR-based and IHC-based assessments of ER and PgR showed good concordance. Both low tumour MKI67 mRNA (RT-qPCR) and Ki-67 protein (IHC) levels were prognostic for favourable DDFS [hazard ratio (HR) 0.42, 95 % CI 0.25-0.71, P = 0.001; and HR 0.56, 0.37-0.84, P = 0.005, respectively] and OS. In multivariable analyses, cancer MKI67 mRNA content had independent influence on DDFS (adjusted HR 0.51, 95 % CI 0.29-0.89, P = 0.019) while Ki-67 protein expression had not any influence (P = 0.266) whereas both assessments influenced independently OS. Luminal B patients treated with docetaxel-FEC had more favourable DDFS and OS than those treated with vinorelbine-FEC when the subtype was defined by RT-qPCR (for DDFS, HR 0.52, 95 % CI 0.29-0.94, P = 0.031), but not when defined using IHC. Breast cancer subtypes approximated with RT-qPCR and IHC show good concordance, but cancer MKI67 mRNA content correlated slightly better with DDFS than Ki-67 expression. The findings based on MKI67 mRNA content suggest that patients with luminal B cancer benefit more from docetaxel-FEC than from vinorelbine-FEC.
  • Xhelilaj, Kaltra (Helsingin yliopisto, 2021)
    Potyviruses are positive-sense single-stranded RNA viruses that can alter several functions of their host plants and consequently, cause significant economic losses in the infected crop plants. During the viral infection, the host transcriptome changes. Stress related genes are triggered, and genes allowing for susceptibility are target for viral-induced modifications. Therefore, in this study, we investigated whether the expression of potential proviral genes SUO1, AGO1, and the major antiviral player AGO2 change in Nicotiana benthamiana (N. benthamiana) in response to potato virus A (PVA; genus Potyvirus) infection. Moreover, we aimed to determine whether helper component protease (HCPro) and active replication have a role in the transcriptional regulation of these genes. Leaves infected with PVA tagged with Renilla luciferase were collected at 3, 6, and 9 days postinoculation, and the viral gene expression was quantified with a dual-luciferase assay. Total RNA was isolated, cDNA was synthesized, and samples were analyzed through qPCR. BLAST hit results revealed that N. benthamiana has three homologs of the SUO1 gene. qPCR data showed no significant change in neither the expression of SUO1 homologs nor the expression of AGO1 during wild-type PVA infection. Moreover, the lack of HCPro or viral replication did not affect the expression of these genes. On the other hand, the expression of AGO2 was approximately 6 and 5 fold up-regulated at 6 and 9 days post-inoculation, respectively. In contrast with the wild-type PVA infection, the mutated viruses had a pronounced effect on AGO2 transcripts at 3 days post-inoculation. Replication-deficient viral RNA increased AGO2 expression circa four-fold, followed by the HCPro-deficient viral RNA increasing expression circa two-fold. AGO2, the major player involved in antiviral defense, was up-regulated during the wild-type infection. Active viral replication and functional HCPro played a role in AGO2 regulation. However, Agrobacterium infiltration can be accounted for interfering with the interpretation of the AGO2 results. Although SUO1 and AGO1 may be potential genes allowing for susceptibility, this study revealed that the PVA infection does not affect the mRNA expression of these genes. Furthermore, it is concluded that active HCPro and viral replication do not have a role in the expression of these genes on mRNA level. To have a clearer view, integrating small RNA, mRNA, and protein quantification analysis of SUO1 homologs will be necessary. Keywords
  • Paju, Susanna; Tallgren, Marika; Kivimäki, Anne; Lahdentausta, Laura; Salminen, Aino; Oksanen, Lotta-Maria Adele Helena; Sanmark, Enni; Geneid, Ahmed; Pussinen, Pirkko; Pietiäinen, Milla (2022)
    Saliva is an alternative sample material to nasopharyngeal swab in SARS- CoV- 2 diagnostics. We investigated possible aspects to improve the reliability of SARS- CoV- 2 detection from saliva. Saliva was collected from asymptomatic healthy subjects (n=133) and COVID- 19 patients (n=9). SARS- CoV- 2 detection was performed with quantitative reverse- transcriptase PCR (RT- qPCR) with two viral and one host target serving as an internal control. The use of internal control revealed that in the first RT- qPCR run 25???30 % of assays failed. The failure is associated with poor RNA quality. When the amount of RNA was cut down to half from the original amount, the performance of RT- qPCR was greatly enhanced (95 % of the assays succeeded). The quality of RNA was not affected by the use of different nucleic acid stabilizing buffers. Our study showed that saliva is suitable material for RT- qPCR based SARS- CoV- 2 diagnostics, but the use of internal control is essential to distinguish the true negative samples from failed assays.
  • Muranen, Sampo (Helsingin yliopisto, 2019)
    Tree shoot architecture research is important due to its significance in fields such as timber production, fruit and nut production and aesthetics of common areas. Also, research on genetic factors that regulate shoot and root system architecture might provide novel methods to store more carbon in forests and, hence, mitigate global warming in the future. LAZY1 is one of the major genes that affects branch and tiller angle in herbaceous and woody species such as Arabidopsis, rice and peach tree. LAZY1 has been under scrutiny over a decade but its molecular function remains unknown. However, it is known that lazy1 mutation affects polar auxin transport. Here it is studied how LAZY1 affects initial branch angle, fiber length and reaction wood development in silver birch (Betula pendula). Also, transcript levels of few shoot architecture related genes were analyzed. LAZY phylogenetic analysis provided evidence of a duplication of LAZY1 in three studied tree species (Betula pendula, Prunus persica, Populus trichocarpa), duplicated genes are here named LAZY1a and LAZY1b. Plant material employed in this study was a segregating population (50:50) of back-cross 1 of weeping birch (B. pendula ´Youngii´) which has a truncated lazy1a. Histological samples of branches were prepared by cryo-sectioning, stained with carbohydrate binding Alcian Blue and lignin binding Safranin dyes to reveal patterns of tension wood development. Due to the large size of branch sections, samples were imaged with a microscope and the images were merged together in a Photoshop application. Branch angles were measured manually with a protractor (angle) tool from stem to the middle of a branch. The data was analyzed using mixed linear models due to the nature of used plant material. We could not use clones because of major issues in in vitro propagation. Branch samples were macerated, fibers imaged and measured by ImageJ software. LAZY1a gene expression levels were analyzed by RT-qPCR method. RNA-sequence analysis indicated that the expression pattern of LAZY1a and LAZY1b is similar in B. pendula. However, one should construct a promoter-reporter line to study with better resolution if their expression is spatially analogous. Initial branch angle was significantly different in wild type compared to lazy1a mutant. For future, one could generate single and double knock out lines of lazy1a/b to study if they have cumulative effect on the branch angle, an important factor in timber quality. Tension wood formation was difficult to quantify with the employed method, due to issues in segregating G-layered tension wood from thick-walled reaction wood. A chemical analysis of cellulose content might provide a more objective method to observe tension wood in branches. RT-qPCR method indicated that LAZY1a transcript levels are higher in wild type compared to mutant. A complementation or knock down experiment would provide sound evidence that lazy1a induces the weeping phenotype. X-ray diffraction method could be employed to study the orientation of cellulose microfibril angle in branches of the wild type vs. mutant. Generation of effective tensional stress requires a cellulose microfibril angle less than 10 and this angle is affected by auxin concentration. It is possible, that this angle is larger in lazy1a due to defect in polar auxin transport.
  • Huusari, Noora (Helsingin yliopisto, 2020)
    Social insects such as ants live in societies and have a strict division of labor between reproductive and worker castes. A colony can consist of even millions of individuals and the number of queens can vary a lot. Populations where each colony comprises just one or few queens are often called kin structured because the relatedness between nestmates is high. Colonies that have lots of queens and the society lives in many connected nests (polydomy) in are referred to as supercolonies. In these colonies relatedness between individuals is low and the workers represent many genetic lineages. Depending on species and the environment where the colony lives societies can behave aggressively towards individuals from other nests to protect their own nest. Ants must be able to recognize members of their own colony from the intruders to be able to protect the nest. Nestmate recognition is a key element in the interaction between nests and species and makes it possible for the workers in the colony to favour their own nestmates in form of care, defence or food acquisition to gain inclusive fitness benefits. To recognise nestmates ants must be able to sense chemical cues. Ants detect these chemical signals through the proteins expressed mainly in their antennas. In this thesis I studied gene expression of genes related to chemosensation in seven Formica species using the RT qPCR method. My study species were kin structured Formica exsecta, F. pratensis and F. fusca and supercolonial F. truncorum, F. pressilabris, F. cinerea and F. aquilonia. My study genes belong to gene families that code for odorant binding proteins (OBP), chemosensory proteins (CSP) and gustatory reseptors (GRT). I want to find out whether the expression of these genes differs between castes, and whether the caste difference varies between kin structured and supercolonial species. Workers have many tasks in the ant colony and to take care of them, they need to have a sophisticated sensory system. For that reason, I expect to find out that the study genes are expressed more in the worker than the queen caste. In addition, I expect the caste difference in gene expression to be higher in the kin structured species than in the supercolonial species. That is because kin structured species behave more aggressively towards intruders and possibly confront intruders more often than the individuals living in supercolonies. Furthermore, in the supercolonies low relatedness between individuals sometimes lead to conflicts inside the nest. For that reason, I suppose queens of the supercolonies express chemosensory genes more than the queens from the kin structured colonies. Overall expression level was the highest for the OBP and the lowest for GRT. The expression level of CSP was in between these extremes. In accordance with my hypothesis gene expression of OBP and CSP was higher in workers in all the study species. GRT expression was worker biased in six of the seven species. Caste difference in expression of chemosensory genes was similar in kin structured and supercolonial species. The expression level varied between species but did not show a pattern depending on the degree of the polygyny. The study revealed that the expression of OBP and CSP is correlated. My results revealed expected worker biased pattern in the expression. The result might be a consequence of better olfactory or taste abilities in the worker caste compared to queens or it may even be consequence of more sophisticated nestmate recognition skills of the workers. This study reveals valuable information about the gene expression of chemosensory genes related to the recognition system in the ants and awakes many new study questions. Chemical sensory system has been studied a lot in the ants, but in the field of expression studies there is still lot to reveal.
  • Shrestha, Rashmi (Helsingin yliopisto, 2018)
    Inorganic phosphate (Pi) is the only readily utilizable form of phosphorus for toxic diazotrophic cyanobacterium Nodularia spumigena (N. spumigena). Pi is one of the limiting nutrients in the Baltic Sea where surprisingly N. spumigena are highly abundant especially during the summer. This indicates that N. spumigena possibly has an alternative pathway to fulfill its phosphorus requirement. The Baltic Sea, like most aquatic environments, is enriched with organic phosphorus compounds among which phosphonates may constitute a significant fraction. Interestingly, the Baltic Sea N. spumigena strains UHCC 0039 and CCY9414 have been found to carry phosphonate degrading gene cluster (phnC-M) implying that these cyanobacteria could assimilate phosphonates as a phosphorus source. However, the significance of the presence of phn gene cluster in N. spumigena for phosphonate utilization has not been investigated in detail. Here, I aimed to understand how N. spumigena copes with Pi limitation and utilizes phosphonates in laboratory conditions using biochemical assays, PCR-based methods and bioinformatics tools. This would aid in finding a suitable marker for Pi deficiency in cyanobacterial blooms in the Baltic Sea. In this study, bioinformatics and PCR screening showed that phn gene cluster was conserved in the Baltic Sea N. spumigena strains. The studied N. spumigena strains UHCC 0039 and UHCC 0060 were found to utilize naturally produced low molecular weight phosphonates, methylphosphonate (MPn), ethylphosphonate (EPn) and 2-aminoethylphosphonate (2APn). Among these phosphonates, MPn seemed to be the most preferred phosphorus source. Alkaline phosphatase activity, an indicator of Pi limitation, was found to be elevated in the media with Pi and 2APn questioning its suitability as a marker for phosphorus limitation. In addition, growth on MPn released methane indicating that massive blooms of N. spumigena might contribute to an elevated methane supersaturation in the Baltic Sea. Reverse transcriptase quantitative PCR (RT-qPCR) in N. spumigena strains did not show expected upregulation of high-affinity phosphate transporter pstS in Pi limitation. It demonstrated an induction of phosphonate transporter gene phnD in media lacking Pi and supplemented by 2APn. The phosphonate lyase gene phnJ was however, upregulated only in the presence of MPn suggesting that phnJ gene could be used as a marker for phosphonate bioavailability. The findings from this study suggest that the presence of phn gene cluster could provide N. spumigena a competitive advantage in Pi-limited cyanobacterial blooms in the Baltic Sea. The molecular detection methods designed in this study thus could be used in future to monitor the expression of genes induced during Pi limitation and the presence of phosphonates, and the method could be further optimized for screening natural water samples.
  • Paavola, Katja (Helsingin yliopisto, 2017)
    Endophytic bacterial strain Serratia plymuthica A30 has been shown to be a good bio-control candidate against pathogen Dickeya solani that is causing soft-rot of potato (Solanum tuberosum) also in Finland. The aim of this master’s thesis was to analyze previously produced RNA sequencing data and to understand which hormone signaling pathways were mainly activated in potato tubers treated by antagonist Serratia plymuthica A30 and Dickeya solani Ds 432-1. The main objective was to compare transcriptional differences of the genes related to jasmonate/ethylene signaling pathways and salicylic acid signaling pathway in pathogen/antagonist treated potato and pathogen treated potato at 24 hour time point after treatments. The additional objective was to identify potential differences between gene groups related to plant defense responses. RNA sequencing data was verified by RT-qPCR method. According to the RNA sequencing, the most active hormonal signaling pathways were jasmonate, ethylene and brassinosteroid signaling pathways after simultanous treatment with pathogen and antagonist. The least active pathways were salicylic acid and gibberellin signaling pathways. However, salicylic and gibberellic acid pathways were the most active in pathogen treated potato tubers and jasmonate, ethylene, auxin and brassinosteroid signaling pathways showed less activity. Based on the results of this thesis, it would appear that the high efficiency of Serratia plymuthica A30 against Dickeya solani pathogen in potato would be based on its direct inhibiting effect against the pathogen but also on the possible activation of jasmonate/ethylene signalling pathway which may lead to an efficient defense response against necrotrophic pathogens.
  • Lee, Hyejeong (Helsingfors universitet, 2014)
    Quantitative reverse transcription PCR (RT-qPCR) assay is widely used for the detection of RNA viruses in environmental water samples. However, a major limitation of using RT-qPCR assay to quantify virus titers is its inability to discriminate between infectious and non-infectious viruses, resulting in overestimation of viral infectivity. Thus, the aim of this study was to develop a reliable molecular method for rotavirus detection with information on viral infectivity, and which may contribute to the development of molecular detection methods for correct estimation of infectivity of non-cultivable viruses. In experimental work, the potential of using propidium monoazide (PMA) or RNase treatment prior to RT-qPCR assay was evaluated to measure the infectivity of human rotavirus. In brief, original human rotavirus (HRV) stock was produced by propagating viruses in MA-104 cells. The virus stocks (including HRV stock A and B) were thermally treated at 80 °C at different time points. The virus titer was measured by (1) cell culture-based infectivity assay, (2) RT-qPCR assay, and (3) RT-qPCR assay with PMA or RNase pretreatment. The result of cell culture-based infectivity assay showed that heat exposure for 5 min at 80 °C was sufficient to inactivate the HRV, while RT-qPCR assay alone overestimated the viral infectivity. The results of RT-qPCR assay with pre-treatments showed that, for thermally-inactivated HRV stock A, similar level of false-positive results was reduced with PMA treatment regardless of inactivation time (ranges from 1.04 to 1.18 log10 PCR-units), while higher reduction level was observed with RNase treatment (ranges from 2.64 to 2.89 log10 PCR-units). On the other hand, the effects of both pre-treatments on thermally-inactivated HRV stock B were negligible. In conclusion, both PMA and RNase pre-treatments eliminated the false-positive results of RT-qPCR assay to some extent in defined conditions, while the discrepancy between the infectivity assay and RT-qPCR assay even with PMA or RNase treatment was observed. In order to confirm the potential of using RT-qPCR assay combined with pre-treatments to measure the infectivity of rotavirus, further studies on optimization of PMA and RNase treatments and production of optimal virus stock would be necessary.
  • Mikkonen, Venla (Helsingin yliopisto, 2022)
    Norovirus on merkittävä ruuansulatuskanavan patogeeni, joka aiheuttaa ihmisillä vuosittain infektioita ympäri maailman. Norovirukset voidaan jaotella kymmeneen eri genoryhmään, joissa on pääasiassa lajispesifisiä infektion aiheuttajia. Genoryhmän II norovirukset aiheuttavat infektioita sekä sioille että ihmisille, mutta pääasiassa sian ja ihmisen norovirukset ovat kuitenkin eri genotyyppejä. Mielenkiinnon on herättänyt kyseisten genotyyppien geneettinen samankaltaisuus ja norovirustestien lajispesifisyys, sillä joissakin tutkimuksissa on todettu ihmisen norovirustestin antaneen positiivisia tuloksia sian noroviruksille. Noroviruksen 15216-2-ISO-standardin mukaisen genoryhmän II PCR (eng. polymerase chain reaction)-testin tavoitteena on havaita noroviruksen genomi riskielintarvikkeista, kuten marjoista ja hedelmistä. Pääsääntöisesti testi on suunniteltu havaitsemaan ihmisten noroviruksia. Tässä alkuperäistutkimuksessa keskityttiin selvittämään noroviruksen 15216-2-ISO-standardin noroviruksen genoryhmän II mukaisten alukkeiden lajispesifisyyttä reaaliaikaisella RT-PCR (eng. reverse transcriptase polymerase chain reaction)-testillä, sillä aiempien tutkimuksien tulokset ovat herättäneet jatkotutkimuksen tarpeen. Ennen tutkimuksen tekoa uskoimme ristireaktioiden olevan mahdollisia genoryhmän II sian ja ihmisen koroviruksien samankaltaisen genomin vuoksi. Tutkimuksemme on tietääksemme ensimmäinen, jossa tutkitaan ihmisen genoryhmän II noroviruksille säädetyn 15216-2-ISO-standardin lajispesifisyyttä sian noroviruksen suhteen. Tutkimuksessa käytimme pakastettuja ja tuoreita sian ulostenäytteitä. Tutkimme yhteensä 100 testattavaa ulostenäytettä, yhden sian norovirukselle positiiviseksi todetun ulostenäytteen sekä 11 sian ulostetta sisältävää jätevesinäytettä. Teimme ulosteista ulostesuspensiot ja eristimme suspensioista RNA:ta kaupallisen eristyssarjan avulla. Tämän jälkeen monistimme näytteessä mahdollisesti olevaa noroviruksen RNA:ta DNA:na reaaliaikaisen käänteiskopioinnin ja polymeraasiketjureaktion avulla. Tutkimme näytteitä kahdessa erilaisissa testiolosuhteissa. 15216-2-ISO-standardin alukkeiden lisäksi testasimme positiivisen tuloksen antaneet ulostenäytteet ja kolme voimakkaimman tuloksen antanutta sian ulostenäytettä sisältävää jätevesinäytettä COG2R ja COG2F -alukkeilla. Saimme tutkimuksessamme 15216-2-ISO-standardin mukaisilla alukkeilla positiivisen tuloksen sian norovirukselle jo aiemmin sian norovirukselle positiiviseksi todetusta sian ulostenäytteestä. Lisäksi saimme positiivisia tuloksia seitsemästä muusta sian ulostenäytteestä ja yhdeksästä sian ulostetta sisältävistä jätevesinäytteistä kyseisillä alukkeilla. Kaikki seitsemän positiivisen tuloksen antanutta sian ulostenäytettä antoivat positiivisen tuloksen myös COG2R ja COG2F -alukkeilla. Kolme kyseisillä alukkeilla testaamaamme sian ulostetta sisältävää jätevesinäytettä antoivat positiivisen tuloksen. Tuloksemme tukevat ajatustamme siitä, että 15216-2-ISO-standardin mukainen RT-qPCR-testi havaitsee sian noroviruksia, sillä tutkimuksemme osoitti, että 15216-2-ISO-standardin mukainen RT-qPCR-testi voi antaa positiivisen tuloksen ainakin yhdelle sian noroviruksen genotyypille. Tutkimuksemme perusteella emme vielä osaa sanoa, antaako testi positiivisen tuloksen kaikille sian noroviruksen genoryhmän II genotyypeille, joten jatkotutkimuksissa positiivisen tuloksen antaneet näytteet sekvensoidaan tai muulla tavoin varmistetaan sian noroviruksiksi.
  • Rytkönen, Annastiina; Tiwari, Ananda; Hokajärvi, Anna Maria; Uusheimo, Sari; Vepsäläinen, Asko; Tulonen, Tiina; Pitkänen, Tarja (2021)
    For microbial source tracking (MST), the 16S ribosomal RNA genes (rDNA) of host-specific bacteria and mitochondrial DNA (mtDNA) of animal species, known to cause fecal contamination of water, have been commonly used as molecular targets. However, low levels of contamination might remain undetected by using these DNA-based qPCR assays. The high copy numbers of ribosomal RNA (rRNA) could offer a solution for such applications of MST. This study compared the performance of eight MST assays: GenBac3 (general Bacteroidales), HF183 (human), BacCan (dog), Rum-2-Bac (ruminant), Pig-2-Bac (swine), Gull4 (gull), GFD, and Av4143 (birds) between rRNA-based and rDNA-based approaches. Three mtDNA-based approaches were tested: DogND5, SheepCytB, and HorseCytB. A total of 151 animal fecal samples and eight municipal sewage samples from four regions of Finland were collected for the marker evaluation. The usability of these markers was tested by using a total of 95 surface water samples with an unknown pollution load. Overall, the performance (specificity, sensitivity, and accuracy) of mtDNA-based assays was excellent (95-100%), but these markers were very seldom detected from the tested surface water samples. The rRNA template increased the sensitivity of assays in comparison to the rDNA template. All rRNA-based assays (except Av4143) had more than 80% sensitivity. In contrast, only half (HF183, Rum-2-Bac, Pig-2-Bac, and Gull4) of rDNA-based assays reached this value. For markers targeted to bird feces, the use of the rRNA-based assay increased or at least did not change the performance. Regarding specificity, all the assays had >95% specificity with a DNA template, except the BacCan assay (71%). While using the RNA template for the assays, HF183 and BacCan exhibited only a low level of specificity (54 and 55%, respectively). Further, the HF183 assay amplified from multiple non-targeted animal fecal samples with the RNA template and the marker showed cross-amplification with the DNA template as well. This study recommends using the rRNA-based approach for MST assays targeting bird fecal contamination. In the case of mammal-specific MST assays, the use of the rRNA template increases the sensitivity but may reduce the specificity and accuracy of the assay. The finding of increased sensitivity calls for a further need to develop better rRNA-based approaches to reach the required assay performance.
  • Jääskeläinen, Anne J.; Korhonen, Essi M.; Huhtamo, Eili; Lappalainen, Maija; Vapalahti, Olli; Kallio-Kokko, Hannimari (2019)
    The laboratory confirmation of Zika virus (ZIKV) infection, and the differential diagnosis from other flavivirus infections such as dengue virus (DENV), often requires the use of several diagnostic test types. Cross-reactions and secondary infections complicate the serological diagnosis and specific viral RNA detection assays are often needed for confirming the diagnosis. The aim of this study was to validate serological and molecular methods for diagnosing ZIKV infection. This included the evaluation of a ZIKV RT-qPCR assay for diagnostics that was previously set up for research use and to compare the ZIKV, DENV and TBEV EIA methods. External and in-house controls and pre-characterized sample panels were tested, and also automated and manual nucleic acid extraction methods were compared. A total of ten Finnish traveler patients were diagnosed with acute ZIKV infection during 2015-2017 including one suspected dual DENV and ZIKV infection. These samples along with panels of DENV and tick-bome encephalitis virus (TBEV) infections were used to test the cross-reactive properties of ZIKV, DENV and TBEV IgM assays. Additionally, the diagnosed acute ZIKV patient samples were tested using commercially available diagnostic DENV NS1 antigen assay and a ZIKV NS1 antigen assay intended for research use. The ZIKV RT-qPCR assay was demonstrated to be both specific and sensitive (one genome per reaction) and suitable for routine diagnostic use utilizing automated nucleic acid extraction. Of the tested IgM tests the NS1 antigen-based ZIKV IgM (Euroimmun) assay performed with least cross -reactivity with a specificity of 97.4%. The DENV IgM assay (Focus Diagnostics) had specificity of only 86.1%. The results are in line with previous studies and additionally highlight that also acute TBEV patients may give a false positive test result in DENV and ZIKV IgM assays.
  • Sihto, Henna-Maria (Helsingfors universitet, 2008)
    Y. enterocolitica and Y. pseudotuberculosis are the third most common reported zoonoses after Campylobacter and Salmonella. Y. pseudotuberculosis has caused 10 outbreaks in Finland since 1997. The symptoms of yersiniosis include fever, right-sided abdominal pain and diarrhea. Post-infectious complications are common. Refrigeration is the most common modern food preservation method, however, as psychrotrophs, enteropathogenic Yersinia are able to grow at refrigerator temperatures. However, the mechanism behind psychrotrophy is still unknown. The aim of this study was to determine the expression levels of rstB, evgS and the genes coding for the σE- and σS-factors of Y. pseudotuberculosis IP 32953 at 30 °C and after temperature drop to 5 °C. Total RNA had been extracted before and 30 minutes, 3, and 7 hours after the temperature downshift for previously performed DNA microarray gene expression studies. Low temperature had been found to increase the expression levels of these genes in these studies. The gene expression levels were determined using real-time quantitative PCR (RT-qPCR). RNA-samples were reverse transcribed into complementary DNA (cDNA), which was used as a template in the qPCR-reactions. The gene expression levels at different time points were determined by measuring the signal emitted by the fluorescent dye. THE ∆∆Ct-method was used for relative quantification. The 16S rRNA (locus tag YPTB_RNA_102) was used for normalization. The expression levels of all the studied genes were approximately doubled 30 minutes after the cold shock demonstrating the significance of the genes in the immediate cold shock response. The expression levels were at highest 3 hours after the cold shock: 2,4-fold for rstB, 4,4-fold for evgS, 6,6-fold for sigmaE and 5,6-fold for rpoS compared with the expression level before the cold shock. The expression levels of all the four genes were decreased 7 hours after the cold shock illustrating adaptation to the lower temperature. The results confirm the findings of the DNA microarray gene expression experiments done previously in the research group. In the future the knowledge of the genes involved in the cold shock response can be utilized in controlling the growth of Y. pseudotuberculosis in foodstuffs.