Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Strategy to identify and quantify polysaccharide gums in gelled food concentrates
    Grün, C.H. ; Sanders, P. ; Burg, M. van der; Schuurbiers, E. ; Adrichem, L. van; Velzen, E.J.J. van; Roo, N. de; Brunt, K. ; Westphal, Y. ; Schols, H.A. - \ 2015
    Food Chemistry 166 (2015). - ISSN 0308-8146 - p. 42 - 49.
    locust bean gum - polymerase-chain-reaction - guar gum - capillary-electrophoresis - enzymatic determination - starch industry - raw-materials - xanthan gum - identification - additives
    A strategy for the unambiguous identification and selective quantification of xanthan gum and locust bean gum (LBG) in gelled food concentrates is presented. DNA detection by polymerase chain reaction (PCR) showed to be a fast, sensitive, and selective method that can be used as a first screening tool in intact gelled food concentrates. An efficient isolation procedure is described removing components that may interfere with subsequent analyses. NMR spectroscopy enabled the direct identification of xanthan gum and the discrimination between different galactomannans in the isolated polysaccharide fraction. An enzymatic fingerprinting method using endo-ß-mannanase, in addition to being used to differentiate between galactomannans, was developed into a selective, quantitative method for LBG, whereas monosaccharide analysis was used to quantify xanthan gum. Recoveries for xanthan gum and LBG were 87% and 70%, respectively, with in-between day relative standard deviations below 20% for xanthan gum and below 10% for LBG.
    Field Evaluation of a Push-Pull System to Reduce Malaria Transmission
    Menger, D.J. ; Omusula, P. ; Holdinga, M.R. ; Homan, T. ; Carreira, A.S. ; Vandendaele, P. ; Derycke, J.L. ; Mweresa, C.K. ; Mukabana, W.R. ; Loon, J.J.A. van; Takken, W. - \ 2015
    PLoS ONE 10 (2015)4. - ISSN 1932-6203 - 20 p.
    mosquito anopheles-gambiae - lasting insecticidal nets - polymerase-chain-reaction - spatial repellency - carbon-dioxide - trap catches - house entry - funestus - vectors - attractants
    Malaria continues to place a disease burden on millions of people throughout the tropics, especially in sub-Saharan Africa. Although efforts to control mosquito populations and reduce human-vector contact, such as long-lasting insecticidal nets and indoor residual spraying, have led to significant decreases in malaria incidence, further progress is now threatened by the widespread development of physiological and behavioural insecticide-resistance as well as changes in the composition of vector populations. A mosquito-directed push-pull system based on the simultaneous use of attractive and repellent volatiles offers a complementary tool to existing vector-control methods. In this study, the combination of a trap baited with a five-compound attractant and a strip of net-fabric impregnated with micro-encapsulated repellent and placed in the eaves of houses, was tested in a malaria-endemic village in western Kenya. Using the repellent delta-undecalactone, mosquito house entry was reduced by more than 50%, while the traps caught high numbers of outdoor flying mosquitoes. Model simulations predict that, assuming area-wide coverage, the addition of such a push-pull system to existing prevention efforts will result in up to 20-fold reductions in the entomological inoculation rate. Reductions of such magnitude are also predicted when mosquitoes exhibit a high resistance against insecticides. We conclude that a push-pull system based on non-toxic volatiles provides an important addition to existing strategies for malaria prevention.
    Mosquito host preferences affect their response to synthetic and natural odour blends
    Busula, A.O. ; Takken, W. ; Loy, D.E. ; Hahn, B.H. ; Mukabana, W.R. ; Verhulst, N.O. - \ 2015
    Malaria Journal 14 (2015). - ISSN 1475-2875 - 9 p.
    polymerase-chain-reaction - vector anopheles-gambiae - rice irrigation scheme - sensu-stricto diptera - human skin microbiota - treated bed nets - carbon-dioxide - western kenya - plasmodium-falciparum - semifield conditions
    Background The anthropophilic malaria mosquito Anopheles gambiae sensu stricto (hereafter termed Anopheles gambiae) primarily takes blood meals from humans, whereas its close sibling Anopheles arabiensis is more opportunistic. Previous studies have identified several compounds that play a critical role in the odour-mediated behaviour of An. gambiae. This study determined the effect of natural and synthetic odour blends on mosquitoes with different host preferences to better understand the host-seeking behaviour of mosquitoes and the potential of synthetic odour blends for standardized monitoring. Methods Odour blends were initially tested for their attractiveness to An. gambiae and An. arabiensis in a semi-field system with MM-X traps baited with natural and synthetic odours. Natural host odours were collected from humans, cows and chickens. The synthetic odour blends consisted of three or five previously identified compounds released with carbon dioxide. These studies were continued under natural conditions where odour blends were tested outdoors to determine their effect on species with different host preferences. Results In the semi-field experiments, human odour attracted significantly higher numbers of both mosquito species. However, An. arabiensis was also attracted to cow and chicken odours, which confirms its opportunistic behaviour. A five-component synthetic blend was highly attractive to both mosquito species. In the field, the synthetic odour blend caught significantly more An. funestus than traps baited with human odour, while no difference was found for An. arabiensis. Catches of An. arabiensis and Culex spp. contained large numbers of blood-fed mosquitoes, mostly from cows, which indicates that these mosquitoes had fed outdoors. Conclusions Different odour baits elicit varying responses among mosquito species. Synthetic odour blends are highly effective for trapping mosquitoes; however, not all mosquitoes respond equally to the same odour blend. Combining fermenting molasses with synthetic blends in a trap represents the most effective tool to catch blood-fed mosquitoes outside houses, which is essential for understanding outdoor malaria transmission.
    Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review
    Czajkowski, R.L. ; Pérombelon, M.C.M. ; Jafra, S. ; Lojkowska, E. ; Potrykus, M. ; Wolf, J.M. van der; Sledz, W. - \ 2015
    Annals of Applied Biology 166 (2015)1. - ISSN 0003-4746 - p. 18 - 38.
    carotovora subsp atroseptica - fragment-length-polymorphism - plant-pathogenic bacteria - erwinia-chrysanthemi strains - polymerase-chain-reaction - 16s ribosomal-rna - monoclonal-antibodies - genetic diversity - ssp-carotovorum - peel extracts
    The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Efficient and cost-effective detection and identification methods are essential to investigate the ecology and pathogenesis of the SRE as well as in seed certification programmes. The aim of this review was to collect all existing information on methods available for SRE detection. The review reports on the sampling and preparation of plant material for testing and on over thirty methods to detect, identify and differentiate the soft rot and blackleg causing bacteria to species and subspecies level. These include methods based on biochemical characters, serology, molecular techniques which rely on DNA sequence amplification as well as several less-investigated ones.
    First evidence of crayfish plaque agent in populations of the marbled caryfish (Procambarus fallux forma virginalis)
    Keller, N.S. ; Pfeiffer, M. ; Roessink, I. ; Schulz, R. ; Schrimpf, A. - \ 2014
    Knowledge and Management of Aquatic Ecosystems (2014)414. - ISSN 1961-9502 - 8 p.
    pathogen aphanomyces-astaci - polymerase-chain-reaction - north-american crayfish - fresh-water crayfish - noble crayfish - marmorkrebs decapoda - adaptation - diversity - hagen - cambaridae
    The introduction of non-indigenous species and associated diseases can cause declines in indigenous flora and fauna and threaten local biodiversity. The crayfish plague pathogen (Aphanomyces astaci), carried and transmitted by latent infected North American crayfish, can lead to high mortalities in indigenous European crayfish populations. Although the parthenogenetic marbled crayfish (Procambarus fallax (Hagen, 1870) forma virginalis) is common in the aquarium trade and has established wild populations in Europe, its carrier status is still unknown. This study investigated one captive and three established wild-living marbled crayfish populations for an infection with the crayfish plague pathogen applying real-time PCR. We demonstrate that captive, as well as two wild marbled crayfish populations were infected by A. astaci. Although infection status in laboratory kept specimens reached high levels, marbled crayfish showed no obviously plague-related mortality. Furthermore, sequence analysis revealed that captive crayfish carried the A. astaci genotype Pc, which has earlier been isolated from the North American red swamp crayfish (Procambarus clarkii). The results indicate that due to its positive carrier status marbled crayfish poses a greater threat to local biodiversity in Europe than considered until now.
    Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the international space station: a challenge for rapid and easy molecular detection of virulent B. anthracis.
    Tongeren, S.P. van; Roest, H.I.J. ; Degener, J.E. ; Harmsen, H.J.M. - \ 2014
    PLoS ONE 9 (2014)9. - ISSN 1932-6203
    polymerase-chain-reaction - toxin genes - identification - purification - sequence - strains - gyrb
    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.
    Survey of the crayfisch plague pathogen presence in the Netherlands reveals a new Aphanomyces astaci carrier.
    Tilmans, M.H.J. ; Mrugala, A. ; Svoboda, J. ; Engelsma, M.Y. ; Petie, M. ; Soes, D.M. ; Nutbeam-Tuffs, S. ; Oidtmann, B. ; Roessink, I. ; Petrusek, A. - \ 2014
    Journal of Invertebrate Pathology 120 (2014). - ISSN 0022-2011 - p. 74 - 79.
    north-american crayfish - polymerase-chain-reaction - fresh-water crayfish - persistent infection - central-europe - populations - prevalence - agent
    North American crayfish species as hosts for the crayfish plague pathogen Aphanomyces astaci contribute to the decline of native European crayfish populations. At least six American crayfish species have been reported in the Netherlands but the presence of this pathogenic oomycete with substantial conservational impact has not yet been confirmed in the country. We evaluated A. astaci prevalence in Dutch populations of six alien crustaceans using species-specific quantitative PCR. These included three confirmed crayfish carriers (Orconectes limosus, Pacifastacus leniusculus, Procambarus clarkii), two recently introduced but yet unstudied crayfish (Orconectes cf. virilis, Procambarus cf. acutus), and a catadromous crab Eriocheir sinensis. Moderate levels of infection were observed in some populations of O. limosus and P. leniusculus. Positive results were also obtained for E. sinensis and two Dutch populations of O. cf. virilis. English population of the latter species was also found infected, confirming this taxon as another A. astaci carrier in European waters. In contrast, Dutch P. clarkii seem only sporadically infected, and the pathogen was not yet detected in P. cf. acutus. Our study is the first confirmation of crayfish plague infections in the Netherlands and demonstrates substantial variation in A. astaci prevalence among potential hosts within a single region, a pattern possibly linked to their introduction history and coexistence.
    RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication
    Feenstra, F. ; Gennip, H.G.P. van; Water, S.G.P. van de; Rijn, P.A. van - \ 2014
    PLoS ONE 9 (2014)3. - ISSN 1932-6203
    polymerase-chain-reaction - intragenic recombination - insect cells - protein ns2 - fever virus - vp6 protein - viral-rna - segment - binding - core
    Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges. Obviously, several steps in the Reoviridae family replication cycle require virus specific as well as segment specific recognition by viral proteins, but detailed processes in these interactions are still barely understood. Recently, we have shown that expression of NS3 and NS3a proteins encoded by genome segment 10 of bluetongue virus is not essential for virus replication. This gave us the unique opportunity to investigate the role of RNA sequences in the segment 10 open reading frame in virus replication, independent of its protein products. Reverse genetics was used to generate virus mutants with deletions in the open reading frame of segment 10. Although virus with a deletion between both start codons was not viable, deletions throughout the rest of the open reading frame led to the rescue of replicating virus. However, all bluetongue virus deletion mutants without functional protein expression of segment 10 contained inserts of RNA sequences originating from several viral genome segments. Subsequent studies showed that these RNA inserts act as RNA elements, needed for rescue and replication of virus. Functionality of the inserts is orientation-dependent but is independent from the position in segment 10. This study clearly shows that RNA in the open reading frame of Reoviridae members does not only encode proteins, but is also essential for virus replication
    A high-throughput method for GMO multi-detection using a microfluidic dynamic array
    Brod, F.C.A. ; Dijk, J.P. van; Voorhuijzen, M.M. ; Dinon, A.Z. ; Guimarães, L.H.S. ; Scholtens, I.M.J. ; Arisi, A.C.M. ; Kok, E.J. - \ 2014
    Analytical and Bioanalytical Chemistry 406 (2014)5. - ISSN 1618-2642 - p. 1397 - 1410.
    genetically-modified maize - real-time pcr - polymerase-chain-reaction - modified organisms - reference molecules - quantitative pcr - screening-assay - digital pcr - zea-mays - validation
    The ever-increasing production of genetically modified crops generates a demand for high-throughput DNAbased methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis byDNA basedmethods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.
    Microbial diversity and dynamics of microbial communities during black-slop soaking of soybeans as determined by PCR-DGGE and molecular cloning
    Yan, Y.Z. ; Wolkers-Rooijackers, J.C.M. ; Nout, M.J.R. ; Han, B.Z. - \ 2013
    World Journal of Microbiology and Biotechnology 29 (2013)10. - ISSN 0959-3993 - p. 1969 - 1974.
    gradient gel-electrophoresis - 16s ribosomal-rna - polymerase-chain-reaction - tempe production - temperature - water
    Tempe is a traditional fermented food in Indonesia. The manufacture process is quite complex, which comprises two stages, preparatory soaking of soybeans and fungal solid state fermentation. Daily addition of previous soak water (back-slopping) during the soybean soaking step is considered to be crucial in the manufacture of high quality tempe. The microbial diversity and dynamics of the microbial communities evolving during back-slop soaking of soybeans for tempe making was investigated by culture-independent PCR–DGGE and molecular cloning. Both DNA and total RNA were isolated and included in this study, to obtain a view on the succession of total and viable bacteria in the complex microbiota. DGGE profiles indicated that Enterobacter sp., Enterococcus sp., Pseudomonas putida, Leuconostoc fallax, Pediococcus pentosaceus, and Weissella cibaria, were the predominant bacteria. Their occurrence shifted dramatically during the back-slop soaking procedure. This study combined with previous culture-dependent studies could gain a better understanding of the complex microbiota of traditional fermented food and give useful information for its quality control.
    Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay
    Graaf-van Bloois, L. van der; Bergen, M.A.P. van; Wal, F.J. van der; Boer, A.G. de; Duim, B. ; Schmidt, T. ; Wagenaar, J.A. - \ 2013
    Journal of Microbiological Methods 95 (2013)1. - ISSN 0167-7012 - p. 93 - 97.
    polymerase-chain-reaction - 16s ribosomal-rna - amplified fragment - differentiation - venerealis - strains - samples - jejuni - genes - coli
    Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.
    Animal Botulism Outcomes in the AniBioThreat Project. Biosecur. Bioterror
    Woudstra, C. ; Tevell Aberg, A. ; Skarin, H. ; Anniballi, F. ; Medici, D. De; Bano, L. ; Koene, M.G.J. ; Löfström, Ch. ; Hansen, T. ; Hedeland, M. ; Fach, P. - \ 2013
    Biosecurity and Bioterrorism: biodefense strategy, practice and science 11 (2013)Suppl. 1. - ISSN 1538-7135 - p. S177 - S182.
    real-time pcr - polymerase-chain-reaction - neurotoxin-producing clostridia - mass-spectrometry - quantitative detection - bovine samples - wound botulism - sybr green - group-iii - types c
    Botulism disease in both humans and animals is a worldwide concern. Botulinum neurotoxins produced by Clostridium botulinum and other Clostridium species are the most potent biological substances known and are responsible for flaccid paralysis leading to a high mortality rate. Clostridium botulinum and botulinum neurotoxins are considered potential weapons for bioterrorism and have been included in the Australia Group List of Biological Agents. In 2010 the European Commission (DG Justice, Freedom and Security) funded a 3-year project named AniBioThreat to improve the EU's capacity to counter animal bioterrorism threats. A detection portfolio with screening methods for botulism agents and incidents was needed to improve tracking and tracing of accidental and deliberate contamination of the feed and food chain with botulinum neurotoxins and other Clostridia. The complexity of this threat required acquiring new genetic information to better understand the diversity of these Clostridia and develop detection methods targeting both highly specific genetic markers of these Clostridia and the neurotoxins they are able to produce. Several European institutes participating in the AniBioThreat project collaborated on this program to achieve these objectives. Their scientific developments are discussed here.
    A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories
    Fischer, M. ; Wernike, K. ; Freuling, C.M. ; Müller, T. ; Aylan, O. ; Brochier, B. ; Cliquet, F. ; Vázquez-Morón, S. ; Hostnik, P. ; Huovilainen, A. ; Isaksson, M. ; Kooi, E.A. - \ 2013
    PLoS ONE 8 (2013)3. - ISSN 1932-6203 - 9 p.
    polymerase-chain-reaction - rabies-related viruses - real-time pcr - rt-pcr - cerebrospinal-fluid - saliva samples - viral-rna - bat - assay - infection
    Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory. The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result. Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing.
    Bluetongue virus with mutated genome segment 10 to differentiate infected from vaccinated animals: A genetic DIVA approach
    Rijn, P.A. van; Water, S.G.P. van de; Gennip, H.G.P. van - \ 2013
    Vaccine 31 (2013)44. - ISSN 0264-410X - p. 5005 - 5008.
    polymerase-chain-reaction - serotype - assay
    Bluetongue virus (BTV) includes 24 serotypes and recently even more serotypes are proposed. Mass vaccination campaigns highlight the need for differential diagnostics in vaccinated populations. Bluetongue disease is routinely diagnosed by serological and virological tests by which differentiation infected from vaccinated animals (DIVA principle) is not possible. Real time PCR tests preferably detect all BTV serotypes (panBTV PCR tests). These PCR tests operate as frontline test to detect new BTV incursions. However, highly sensitive panBTV PCR tests can also detect currently applied inactivated and modified-live vaccines. Here, BTV with eight silent mutations in segment 10 (Seg-10) was generated by reverse genetics. This BTV mutant is not detected by a Seg-10 panBTV PCR test (genetic DIVA). Thus, inactivated BT vaccine with this mutated Seg-10 will avoid false positive PCR results post vaccination, whereas BTV infected animals can be positively diagnosed with the accompanying Seg-10 panBTV PCR test (DIVA-test) far beyond the infectious period.
    qPCR Assays for the Detection of Cylindrocladium buxicola in Plant, Water and Air Samples
    Gehesquière, B. ; Haeyer, S. D'; Pham, K.T.K. ; Kuik, A.J. van; Maes, M. ; Höfte, M. ; Heungens, K. - \ 2013
    Plant Disease 97 (2013)8. - ISSN 0191-2917 - p. 1082 - 1090.
    real-time pcr - polymerase-chain-reaction - 1st report - buxus spp. - box blight - specificity - fusarium - belgium - primers - disease
    Cylindrocladium buxicola (syn. C. pseudonaviculatum; teleomorph Calonectria pseudonaviculata) is an important fungal pathogen of Buxus spp. Although widespread in Western Europe, this pathogen has only recently been introduced into North America, where it represents a significant threat to the U.S. and Canadian boxwood industries. Trade of latently infected nursery stock is an important mode of long-distance dissemination and introduction of this pathogen but no methods for detection of latently infected material are available. Also, the pathways for short-distance dispersal of C. buxicola have not been adequately studied. Improved detection methods of this pathogen in air and water samples would benefit future research in this area. We have developed real-time polymerase chain reaction assays for the detection of C. buxicola based on the ribosomal DNA internal transcribed spacer 1 (ITS) and the ß-tubulin 2 gene (TUB). Using a TaqMan probe conjugated with a 3' minor groove binding group (TaqMan MGB probe), the ITS-based assay could reliably detect as little as 10 fg of genomic DNA or 20 copies of cloned target DNA and was approximately 70 times more sensitive than the SYBR Green TUB-based assay. The ITS-based assay provided good but not complete specificity, and is well suited for epidemiological studies. The TUB-based assay, however, proved to be fully specific and can be used for diagnostics. We developed and optimized sample processing and DNA extraction methods for detection of latently present C. buxicola in boxwood plants and quantification of conidia in water and air samples. C. buxicola could be detected in 20 g of plant material, of which only 1 ppm of the tissue was infected, in 10-ml water samples containing as low as 1 conidium/ml, and on Melinex tape pieces representing 12 h of air sampling containing 10 or more conidia. The applicability of the techniques to plant, water, and air samples of practical size was demonstrated.
    Detection of bacterial DNA in bile of cats with lymphocytic cholangitis
    Otte, C.M.A. ; Pérez, O.N. ; Favier, R.P. ; Rothuizen, J. ; Penning, L.C. - \ 2012
    Veterinary Microbiology 156 (2012)1-2. - ISSN 0378-1135 - p. 217 - 221.
    16s ribosomal-rna - gradient gel-electrophoresis - polymerase-chain-reaction - helicobacter-pylori - pcr amplification - sp-nov. - jeotgalicoccus - disease - liver - populations
    In this study, we have successfully used molecular methods based on the amplification of the 16S ribosomal RNA gene on feline bile samples to show that bile of cats with LC is not sterile. This is probably due to the fact that the inflammatory process in the biliary tree causes dilatations. As a result, bacteria can easily migrate from the intestines via the common bile duct. The diversity of species identified and the presence of Helicobacter spp. DNA in both patients and controls suggests that bacteriobilia is secondary to the disease and is not the cause of LC.
    A Universal Microarray Detection Method for Identification of Multiple Phytophthora spp. Using Padlock Probes
    Sikora, K. ; Verstappen, E.C.P. ; Mendes, O. ; Schoen, C.D. ; Ristaino, J. ; Bonants, P.J.M. - \ 2012
    Phytopathology 102 (2012)6. - ISSN 0031-949X - p. 635 - 645.
    polymerase-chain-reaction - internal transcribed spacer - real-time pcr - ribosomal dna - phylogenetic-relationships - natural ecosystems - plant-pathogens - reaction assay - ramorum - quantification
    The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.
    Fine Scale Spatiotemporal Clustering of Dengue Virus Transmission in Children and Aedes aegypti in Rural Thai Villages
    Yoon, I.K. ; Getis, A. ; Aldstadt, J. ; Rothman, A.L. ; Tannitisupawong, D. ; Koenraadt, C.J.M. ; Fansiri, T. ; Jones, J.W. ; Morrison, A.C. ; Jarman, R.G. ; Nisalak, A. ; Mammen Jr., M.P. ; Thammapalo, S. ; Srikiatkhachorn, A. ; Green, S. ; Libraty, D.H. ; Gibbons, R.V. ; Endy, T. ; Pimgate, C. ; Scott, T.W. - \ 2012
    PLoS Neglected Tropical Diseases 6 (2012)7. - ISSN 1935-2727
    polymerase-chain-reaction - primary-school children - kamphaeng phet - puerto-rico - vector - culicidae - diptera - blood - patterns - kinetics
    Background Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages. Methodology/Principal Findings Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile “index” cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (p
    Virus hazards from food, water and other contaminated environments
    Rodriguez-Lázaro, D. ; Cook, N. ; Ruggeri, F.M. ; Sellwood, J. ; Nasser, A. ; Nascimento, M.S. ; Agostino, M. D'; Santos, R. ; Saiz, J.C. ; Rzezutka, A. ; Bosch, A. ; Girones, R. ; Carducci, A. ; Muscullo, M. ; Kovac, K. ; Diez-Valcarce, M. ; Vantarakis, A. ; Bonsdorff, C.H. ; Roda Husman, A.M. de; Hernández, M. ; Poel, W.H.M. van der - \ 2012
    FEMS Microbiology Reviews 36 (2012)4. - ISSN 0168-6445 - p. 786 - 814.
    hepatitis-e-virus - reverse transcription-pcr - human enteric viruses - polymerase-chain-reaction - norwalk-like virus - cell-culture-pcr - time rt-pcr - sequence-based amplification - human pathogenic viruses - treated drinking-water
    Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run-offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the Caliciviridae, Adenoviridae, Hepeviridae, Picornaviridae and Reoviridae. Sampling methods and strategies, first-choice detection methods and evaluation criteria are reviewed.
    The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato
    Gruyter, J. de; Gent-Pelzer, M.P.E. van; Woudenberg, J.H.C. ; Rijswick, P.C.J. van; Meekes, E.T.M. ; Crous, P.W. ; Bonants, P.J.M. - \ 2012
    European Journal of Plant Pathology 134 (2012)2. - ISSN 0929-1873 - p. 301 - 313.
    polymerase-chain-reaction - phoma-tracheiphila - didymella-bryoniae - complex - foveata - differentiation - phylogeny - multiplex - sequences - disease
    Stagonosporopsis andigena and S. crystalliniformis are serious foliage pathogens on potato (Solanum tuberosum) and tomato (Solanum lycopersicum). As both species have been recorded only in the Andes area, S. andigena is listed as an A1 quarantine organism in Europe. The actin region of isolates of Stagonosporopsis and allied species of Boeremia, Didymella, Peyronellaea and Phoma was amplified using generic primers. DNA sequence differences of the actin gene were utilised to develop species-specific real-time (TaqMan) PCR assays for the detection of S. andigena and S. crystalliniformis in leaves of potato or tomato. The specificity of the TaqMan PCR assays was determined on genomic DNA extracted from two S. andigena and two S. crystalliniformis isolates and 16 selected isolates of Stagonosporopsis, Phoma and Boeremia, which are the closest relatives. The validation of the methods developed included the DNA extraction and the TaqMan PCR assays. The performance criteria specificity, analytical sensitivity, reproducibility, repeatability and robustness of the TaqMan PCR assays demonstrated the reliability of both methods for the detection of S. andigena and S. crystalliniformis in leaf material. The TaqMan PCR assays were tested on symptomatic leaves of potato and tomato that were obtained after artificial inoculation of detached leaves with both pathogens under quarantine conditions. In the artificial inoculation experiments both S. andigena and S. crystalliniformis caused leaf infections on potato and tomato.
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