Detection of bovine foetal DNA from amniotic fluid using the polymerase chain reaction

Selostus: Sikiön DNA:n tunnistaminen naudan sikiövedestä polymeraasiketjureaktion avulla

Universally primed polymerase chain reaction analysis of Fusarium avenaceum isolated from wheat and barley in Finland

Selostus: Vehnästä ja ohrasta eristettyjen F. avenaceum -punahomekantojen analysointi UP-PCR-menetelmällä

Aikaerotteisesti fluoresenssia mittaavan reaaliaikaisen PCR-laitteen kehitys

Kvantitatiivinen reaaliaikainen polymeraasiketjureaktio (engl. polymerase chain reaction, PCR) on osoittautunut käyttäjäystävällisimmäksi menetelmäksi nukleiinihapposekvenssien kvantitoimisessa. Tätä menetelmää voidaan herkistää pienempien DNA-pitoisuuksien havaitsemiseen käyttämällä hyväksi aikaerotteista fluorometriaa (engl. time-resolved fluorometry, TRF) ja luminoivia lantanidileimoja, joiden fluoresenssin pitkän eliniän ansiosta emission mittaus voidaan suorittaa vasta hetki virittävän valopulssin jälkeen, jolloin lyhytikäinen taustasäteily ehtii sammua. Tuloksena saadaan korkea signaali-taustasuhde. Tämän diplomityön tarkoituksena oli rakentaa TRF:än pystyvä reaaliaikainen PCR-laite, sillä tällaista laitetta ei ole markkinoilla tarjolla. Laite rakennettiin kehittämällä lämpökierrätin ja yhdistämällä se valmiiseen TRF:än kykenevään mittapäähän. Mittapään ja lämpökierrättimen hallitsemiseksi kehitettiin myös tietokoneohjelma. Valon tuottamiseksi ja mittaamiseksi haluttiin käyttää edullisia komponentteja, joten työssä käytettiin valmiin mittapään optiikkaa, jossa viritys tapahtuu hohtodiodilla (engl. light-emitting diode, LED) ja lantanidileiman emission mittaus fotodiodilla (engl. photodiode, PD) tai valomonistinputkella (engl. photomultiplier tube, PMT). Myös mittapään suorituskykyä tutkittiin. Työtä varten kehitettiin lämpökierrätin, joka koostui Peltier-elementillä lämmitettävästä PCR-putkitelineestä ja lämpökannesta. Mittalaitteen suorituskyvyn tutkimiseen käytettiin kelaattikomplementaatioon perustuvaa PCR-tuotteen havaitsemismenetelmää. Kelaattikomplementaatio perustuu kahteen erilliseen oligonukleotidimolekyyliin, joista toiseen on sidottu lantanidi-ioni ja toiseen valoa absorboiva ligandirakenne, jotka yhdessä muodostavat fluoresoivan kokonaisuuden. Kehitetyn lämpökierrättimen todettiin olevan tarpeeksi tarkka sekä tehokas ja sen lämmitys- ja jäähdytysnopeuden maksimeiksi saatiin 2,6 °C/sekunti. Detektorina käytetyn PD:n ei todettu olevan tarpeeksi herkkä emission havainnoimiseksi ja se korvattiin laitteessa PMT:llä. Käytetyllä PCR-määrityksellä kynnyssykleiksi (engl. threshold cycle, Ct) sekä kehitetylle että referenssilaitteelle saatiin 28,4 käyttämällä samaa 100 000 kopion DNA:n aloitusmäärää. Työssä osoitettiin, että on mahdollista kehittää edullisia komponentteja käyttävä, TRF:än pystyvä, reaaliaikainen PCR-laite, joka kykenee vastaavaan Ct-arvoon kuin vertailulaite. PD:n herkkyys ei kuitenkaan riittänyt. Tulokset olivat lupaavia, sillä LED- ja PD-teknologiat kehittyvät ja markkinoille on tullut myös muita komponentteja, joiden avulla on tulevaisuudessa mahdollista kehittää vielä herkempi laite.

Food Safety Testing: Rapid Molecular Methods for Chemical and Biological Hazards

The central goal of food safety policy in the European Union (EU) is to protect consumer health by guaranteeing a high level of food safety throughout the food chain. This goal can in part be achieved by testing foodstuffs for the presence of various chemical and biological hazards. The aim of this study was to facilitate food safety testing by providing rapid and user-friendly methods for the detection of particular food-related hazards. Heterogeneous competitive time-resolved fluoroimmunoassays were developed for the detection of selected veterinary residues, that is coccidiostat residues, in eggs and chicken liver. After a simplified sample preparation procedure, the immunoassays were performed either in manual format with dissociation-enhanced measurement or in automated format with pre-dried assay reagents and surface measurement. Although the assays were primarily designed for screening purposes providing only qualitative results, they could also be used in a quantitative mode. All the developed assays had good performance characteristics enabling reliable screening of samples at concentration levels required by the authorities. A novel polymerase chain reaction (PCR)-based assay system was developed for the detection of Salmonella spp. in food. The sample preparation included a short non-selective pre-enrichment step, after which the target cells were collected with immunomagnetic beads and applied to PCR reaction vessels containing all the reagents required for the assay in dry form. The homogeneous PCR assay was performed with a novel instrument platform, GenomEra^™, and the qualitative assay results were automatically interpreted based on end-point time-resolved fluorescence measurements and cut-off values. The assay was validated using various food matrices spiked with sub-lethally injured Salmonella cells at levels of 1-10 colony forming units (CFU)/25 g of food. The main advantage of the system was the exceptionally short time to result; the entire process starting from the pre-enrichment and ending with the PCR result could be completed in eight hours. In conclusion, molecular methods using state-of-the-art assay techniques were developed for food safety testing. The combination of time-resolved fluorescence detection and ready-to-use reagents enabled sensitive assays easily amenable to automation. Consequently, together with the simplified sample preparation, these methods could prove to be applicable in routine testing.

Homogeneous Assays for Simplified Screening of HLA-conferred Genetic Disease Risk

The increasing incidence of type 1 diabetes has led researchers on a quest to find the reason behind this phenomenon. The rate of increase is too great to be caused simply by changes in the genetic component, and many environmental factors are under investigation for their possible contribution. These studies require, however, the participation of those individuals most likely to develop the disease, and the approach chosen by many is to screen vast populations to find persons with increased genetic risk factors. The participating individuals are then followed for signs of disease development, and their exposure to suspected environmental factors is studied. The main purpose of this study was to find a suitable tool for easy and inexpensive screening of certain genetic risk markers for type 1 diabetes. The method should be applicable to using whole blood dried on sample collection cards as sample material, since the shipping and storage of samples in this format is preferred. However, the screening of vast sample libraries of extracted genomic DNA should also be possible, if such a need should arise, for example, when studying the effect of newly discovered genetic risk markers. The method developed in this study is based on homogeneous assay chemistry and an asymmetrical polymerase chain reaction (PCR). The generated singlestranded PCR product is probed by lanthanide-labelled, LNA (locked nucleic acid)-spiked, short oligonucleotides with exact complementary sequences. In the case of a perfect match, the probe is hybridised to the product. However, if even a single nucleotide difference occurs, the probe is bound instead of the PCR product to a complementary quencher-oligonucleotide labelled with a dabcyl-moiety, causing the signal of the lanthanide label to be quenched. The method was applied to the screening of the well-known type 1 diabetes risk alleles of the HLA-DQB1 gene. The method was shown to be suitable as an initial screening step including thousands of samples in the scheme used in the TEDDY (The Environmental Determinants of Diabetes in the Young) study to identify those individuals at increased genetic risk. The method was further developed into dry-reagent form to allow an even simpler approach to screening. The reagents needed in the assay were in dry format in the reaction vessel, and performing the assay required only the addition of the sample and, if necessary, water to rehydrate the reagents. This allows the assay to be successfully executed even by a person with minimal laboratory experience.