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Species-specific Primers (species-specific + primer)
Selected AbstractsIdentification of Lophodermium seditiosum and L. pinastri in Swedish forest nurseries using species-specific PCR primers from the ribosomal ITS regionFOREST PATHOLOGY, Issue 3 2005E. Stenström Summary Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species-specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries. Résumé Lophodermium seditiosum est un pathogène important des aiguilles sur pins, particulièrement en pépinières, et il serait nécessaire de détecter le pathogène dans sa phase latente. Les régions ITS de L. seditiosum et L. pinastri ont été amplifiées avec des amorces universelles et séquencées. La comparaison de la séquence des deux espèces a permis de développer des amorces spécifiques pour chaque espèce dans la région ITS. Les amorces ont une longueur de 18 à 24 paires de bases avec un minimum de 3 paires de bases de différence entre espèces. Ces amorces n'ont produit aucune amplification avec l'ADN des autres espèces de champignons testées ou les aiguilles saines de Pinus sylvestris. Il a également été possible de détecter L. seditiosum ou L. pinastri avec ces amorces dans des aiguilles infectées avec ou sans signe d'infection. Cette méthode s'avère très utile pour la détection d'infections latentes de L. seditiosum dans les aiguilles de P. sylvestris en pépinières. Zusammenfassung Lophodermium seditiosum ist ein starkes Nadelpathogen an Kiefern, speziell in Baumschulen. Für den Einsatz von Bekämpfungsmassnahmen wäre es von Vorteil, wenn man das Pathogen bereits während der Latenzperiode nachweisen könnte. Die ITS Regionen der ribosomalen DNA von L. seditiosum und L. pinastri wurden mit Standardprimern amplifiziert und sequenziert. Vergleiche der Sequenzen der beiden Arten erlaubten die Entwicklung von artspezifischen Primern für die ITS Regionen. Die Primerpaare waren zwischen 18 and 24 Basenpaaren lang und wiesen einen Unterschied von mindestens drei Nukleotiden auf. Die DNA von allen anderen untersuchten Pilzarten und von gesunden Pinus sylvestris Nadeln liessen sich mit keinem dieser Primerpaare amplifiziern. Lophodermium seditiosum und L. pinastri konnten mit den Primerpaaren in infizierten Nadeln mit und ohne Symptome direkt nachgewiesen werden. Die Methode eignete sich vorzüglich zum Nachweis von latenten Infektionen von L. seditiosum in P. sylvestris Nadeln in Baumschulen. [source] A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P. citricola in soil samples from oak standsFOREST PATHOLOGY, Issue 2 2001Nechwatal A description is given of the use of a combination of polymerase chain reaction (PCR) and baiting techniques for the specific detection of Phytophthora quercina and Phytophthora citricola from soil around declining oak trees. The soil was flooded with water and subjected to a specific baiting procedure using Quercus robur leaflets as baits. Single round or nested PCR, respectively, with species-specific primers allowed the detection of P. quercina and P. citricola in infected oak leaflets used as baits and in the water from the same bait tests. PCR detection of both fungi was also possible after soil samples had been thoroughly mixed with water and the floating organic debris had been collected. Phytophthora quercina and P. citricola could be readily detected in almost every case in the water from these tests by PCR but less frequently in the organic debris. The identities of P. quercina and P. citricola were confirmed by restriction digests of the corresponding PCR amplicons. The presence of both fungi was also confirmed in parallel in soil samples tested by baiting with oak leaflets. Nested PCR with the primers used allowed the detection of as few as five zoospores of P. citricola and 300 zoospores of P. quercina in a volume of 100 ,l. The methods presented here allow detection and identification of species of Phytophthora in soil without the need for direct extraction of soil samples, and without specific knowledge of the morphological characteristics of the genus. Détection de Phytophthora quercina et de P. citricola dans le sol de chênaies par une méthode combinant le piégeage et la PCR L'article décrit la détection spécifique de Phytophthora quercina et de P. citricola dans le sol prélevé autour de chênes dépérissants, par une méthode combinant les techniques de piégeage et de PCR. Le sol a été immergé dans l'eau et soumis à la procédure du piégeage avec de très jeunes feuilles de Quercus robur. Une amplification par PCR simple ou par PCR gigogne, respectivement, avec des amorces spécifiques des espèces ont permis de détecter P. quercina et P. citricola dans les feuilles-piège infectées, et dans l'eau de piégeage. La détection des deux champignons par PCR a aussi été possible après que les échantillons de sol aient été soigneusement mélangés à de l'eau et que les débris organiques aient été collectés. Phytophthora quercina et P. citricola ont pu être facilement détectés par PCR dans presque tous les cas dans l'eau de piégeage, mais moins fréquemment dans les débris organiques. L'identité de P. quercina et de P. citricola a été confirmée par les profils de restriction des amplifiats obtenus. La présence des deux champignons a aussi été confirmée en parallèle dans des échantillons de sol par piégeage. L'amplification par PCR gigogne avec les amorces utilisées a permis la détection de seulement 5 zoospores de P. citricola et 300 zoospores de P. quercina, dans un volume de 100 ,l. Les méthodes présentées ici permettent la détection et l'identification des espèces de Phytophthora dans le sol en évitant l'extraction directe d'ADN du sol, et sans connaissances spécifiques sur les caractéristiques morphologiques du genre. Eine Kombination von Köder- und PCR-Techniken zum Nachweis von Phytophthora quercina und P. citricola in Bodenproben von Eichenstandorten Es wird der spezifische Nachweis von Phytophthora quercina und P. citricola in Bodenproben von absterbenden Eichen mit Hilfe einer Kombination von PCR- und Baiting-Methoden beschrieben. Die Bodenproben wurden mit Wasser geflutet und Baiting-Tests unterzogen, bei denen junge Blättchen von Quercus robur als Köder zum Einsatz kamen. Einfache oder nested PCR-Reaktionen mit artspezifischen Primern erlaubten den Nachweis von P. quercina und P. citricola in den infizierten Eichenblättchen aus diesen Tests und im jeweiligen ,Baiting-Wasser'. Der PCR-Nachweis beider Erreger war auch möglich, wenn Bodenproben gründlich mit Wasser gemischt wurden, das aufgeschwemmte organische Material abgesammelt und das Wasser abgenommen wurde. P. quercina und P. citricola wurden dabei in nahezu allen Fällen im Wasser, jedoch weniger regelmäßig im organischen Material nachgewiesen. Die Identität der betreffenden Arten wurde zusätzlich durch Restriktions-Analysen der entsprechenden Amplicons bestätigt. Außerdem wurde die Anwesenheit beider Arten in den untersuchten Bodenproben durch klassische Baiting-Methoden nachgewiesen. Nested PCR mit den verwendeten Primerpaaren erlaubte den Nachweis von nur 5 Zoosporen von P. citricola und 300 Zoosporen von P. quercina in einem Gesamt-Volumen von 100 ,l. Die beschriebenen Methoden ermöglichen Nachweis und Identifizierung von Phytophthora-Arten in Bodenproben, ohne die Notwendigkeit einer direkten Extraktion des Bodens und ohne weitreichende Kenntnis der morphologischen Merkmale der Arten dieser Gattung. [source] Stromal MCP-1 in mammary tumors induces tumor-associated macrophage infiltration and contributes to tumor progressionINTERNATIONAL JOURNAL OF CANCER, Issue 6 2009Hiroshi Fujimoto Abstract There is growing evidence that tumor-associated macrophages (TAMs) promote tumor growth and dissemination. Many individual reports have focused on the protumor function of molecules linked to the recruitment of macrophages, but little is known about which factor has the strongest impact on recruitment of macrophages in breast cancer. To elucidate this question, we performed RT-PCR using species-specific primers and evaluated tumoral and stromal mRNA expression of macrophage chemoattractants separately in human breast tumor xenografts. The correlation between the tumoral or stromal chemoattractant mRNA expression including monocyte chemoattractant protein-1 (MCP-1) (CCL2), MIP-1, (CCL3), RANTES (CCL5), colony-stimulating factor 1, tumor necrosis factor ,, platelet-derived growth factor (PDGF)-BB and macrophage infiltration were compared. There was significant positive correlation between stromal MCP-1 expression and macrophage number (r = 0.63), and negative correlation between tumoral RANTES expression and macrophage number (r = ,0.75). However, no significant correlation was found for the other tumoral and stromal factors. The interaction between the tumor cells and macrophages was also investigated. Tumor cell,macrophage interactions augmented macrophage-derived MCP-1 mRNA expression and macrophage chemotactic activity in vitro. Treatment of immunodeficient mice bearing human breast cancer cells with a neutralizing antibody to MCP-1 resulted in significant decrease of macrophage infiltration, angiogenetic activity and tumor growth. Furthermore, immunohistochemical analysis of human breast cancer tissue showed stromal MCP-1 had a significant correlation with relapse free survival (p = 0.029), but tumoral MCP-1 did not (p = 0.105). These findings indicate that stromal MCP-1 produced as a result of tumor,stromal interactions may be important for the progression of human breast cancer and macrophages may play an important role in this tumor,stroma interaction. © 2009 UICC. [source] Chronic shedding of Campylobacter species in beef cattleJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004G.D. Inglis Abstract Aims:, To determine the prevalence of chronic shedding of Campylobacter species by beef cattle, a longitudinal study of shedding patterns was conducted in a cohort of 60 beef steers over a 4-month period. Methods and Results:, Steers were maintained in a simulated feedlot setting but individually in pens to minimize transmission among animals. At each collection time, campylobacters in faeces were detected using conventional PCR. In addition, quantities of Campylobacter jejuni and C. lanienae in faeces were measured using real-time quantitative (RTQ) PCR. All of the steers tested shed Campylobacter species during the course of the study, and overall, 90% of the 299 samples tested were positive for Campylobacter DNA. The majority of the animals (86%) shed campylobacters at ,4 sample times. The most prevalent taxon detected in bovine faeces was C. lanienae (56% of samples) followed by C. jejuni (13%), C. hyointestinalis (8%), and C. fetus (2%). No C. coli was detected, and 13% of the faecal samples contained two or more of the above species. Seven (12%) and 34 (57%) animals shed C. jejuni and C. lanienae at ,3 sample times, respectively. For both C. lanienae and C. jejuni, a substantial number of cells were detected in faeces using RTQ-PCR; 27% of the samples positive for C. jejuni contained populations >104 cells g,1 (maximum of 5 × 105 cells g,1), and 44% of samples positive for C. lanienae possessed populations >106 cells g,1 (maximum of 4 × 108 cells g,1). A significant correlation was observed between shedding of C. lanienae and the severity of liver abscesses. In 27% of the samples, an amplicon was obtained for genus-specific but not for the species-specific primers. Sequencing of the partial 16S rRNA gene suggested the presence of at least two undescribed Campylobacter species but this has yet to be confirmed. Conclusions:, A high percentage of feedlot cattle shed large quantities of Campylobacter species in their faeces over a protracted period of time (ca 112 days). Significance and Impact of the Study:, This is the first study of longitudinal shedding patterns of campylobacters in beef cattle using PCR-detection methods. In addition, this is the first use of RTQ-PCR to directly quantify C. jejuni or C. lanienae in faeces. The results of the study show that a large number of cattle (>85%) chronically shed campylobacters in feedlots. [source] Profiling of subgingival plaque biofilm microflora from periodontally healthy subjects and from subjects with periodontitis using quantitative real-time PCRJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2010Y Abiko Abiko Y, Sato T, Mayanagi G, Takahashi N. Profiling of subgingival plaque biofilm microflora from periodontally healthy subjects and from subjects with periodontitis using quantitative real-time PCR. J Periodont Res 2010; 45: 389,395. © 2010 John Wiley & Sons A/S Background and Objective:, Qualitative and quantitative changes of the subgingival plaque biofilm microflora in periodontal pockets are thought to be associated with the development and progression of periodontitis. The aims of the present study were to quantify the proportions of nine periodontitis-associated bacterial species and four Streptococcus species in subgingival plaque, and to evaluate their relationship with periodontitis quantitatively. Material and Methods:, Subgingival plaque samples were obtained from 12 periodontally healthy subjects and from 28 patients with periodontitis. The amounts of total and target bacteria were measured by quantitative real-time PCR using universal and species-specific primers, respectively. Results:, The proportion of total obligate anaerobes was found to be higher in subjects with periodontitis than in periodontally healthy subjects (p < 0.05). Among obligate anaerobes, Tannerella forsythia (2.04 ± 5.27%, p < 0.05), Porphyromonas gingivalis (0.54 ± 1.41%) and Eubacterium saphenum (0.30 ± 0.96%) were detected at high proportions in subjects with periodontitis, but not in periodontally healthy subjects. By contrast, the proportion of total streptococci was lower in subjects with periodontitis (p < 0.05). Specifically, the proportion of T. forsythia, P. gingivalis or E. saphenum increased (, 2.78%) and the proportion of Streptococcus species decreased to virtually undetectable levels, in subjects with periodontitis. Conclusion:, Obligate anaerobes, including T. forthysia, P. gingivalis and E. saphenum, were identified predominantly in microflora from subjects with periodontitis, whereas Streptococcus species were identified predominantly in microflora from periodontally healthy subjects, suggesting a change in the subgingival environment that resulted in conditions more suitable for the survival of obligate anaerobes. The proportion of these obligate anaerobes in the subgingival plaque of subjects with periodontitis appears to be associated with the status of human periodontitis. [source] Differentiation of Two Pathogens of Powdery Mildew Disease in Flowering Dogwood (Cornus florida) by PCR-mediated Method Based on ITS SequencesJOURNAL OF PHYTOPATHOLOGY, Issue 5 2009Ainong Shi Abstract Two fungi, Phyllactinia guttata and Erysiphe pulchra were identified as the pathogens of powdery mildew of flowering dogwood (Cornus florida). The objective of this research was to identify and distinguish the two fungi by developing species-specific primers. The internal transcribed spacer (ITS) universal primers and a series of species-specific primers designed from the ITS regions were used to evaluate and validate the two fungi causing powdery mildew in dogwood. Four primer pairs showed specificity to P. guttata and three to E. pulchra. These species-specific primer pairs can be used as molecular markers to provide diagnostic tools for detection and differentiation of the two powdery mildew pathogens in flowering dogwood. [source] Use of specific PCR primers to identify three important industrial species of Saccharomyces genus: Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces pastorianusLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2010G.V. De Melo Pereira Abstract Aim:, To develop species-specific primers capable of distinguishing between three important yeast species in alcoholic fermentation: Saccharomyces bayanus, Saccharomyces cerevisiae and Saccharomyces pastorianus. Methods and Results:, Two sets of primers with sequences complementary to the HO genes from Saccharomyces sensu stricto species were used. The use of the ScHO primers produced a single amplificon of c. 400 or 300 bp with species S. cerevisiae and S. pastorianus, respectively. The second pair of primers (LgHO) was also constructed, within the HO gene, composed of perfectly conserved sequences common for S. bayanus species, which generate amplicon with 700 bp. No amplification product was observed in the DNA samples from non- Saccharomyces yeasts. Saccharomyces species have also been characterized via electrophoretic karyotyping using pulsed-field gel electrophoresis to demonstrate chromosomal polymorphisms and to determine the evolutionary distances between these species. Conclusions:, We conclude that our novel species-specific primers could be used to rapidly and accurately identify of the Saccharomyces species most commonly involved in fermentation processes using a PCR-based assay. Significance and Impact of the Study:, The method may be used for routine identification of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes in less than 3 h. [source] Identification and characterization of enterococci from bryndza cheeseLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2006D. Jurkovi Abstract Aims:, To identify enterococci isolated from sheep milk cheese , bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. Methods and Results:, Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylLL, cylLS, cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. Conclusions:,Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. Significance and Impact of the Study:, To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants. [source] Molecular detection of predation by soil micro-arthropods on nematodesMOLECULAR ECOLOGY, Issue 7 2006D. S. READ Abstract The relative importance of the factors driving change in the population dynamics of nematodes in the soil is almost completely unknown. Top-down control by micro-arthropod predators may have a significant impact on nematode population dynamics. We report experiments showing that mites and Collembola were capable of reducing nematode numbers in the laboratory and were feeding on a targeted nematode species in the field. A PCR-based approach was developed for the detection of predation on three species of slug- and insect-pathogenic nematodes: Phasmarhabditis hermaphrodita, Heterorhabditis megidis and Steinernema feltiae. The collembolan Folsomia candida and the mesostigmatid mite Stratiolaelaps miles were employed as model predators to calibrate post-ingestion prey DNA detection times. Fragments of cytochrome oxidase I (COI) mtDNA were sequenced and species-specific primers were designed, amplifying 154-, 154- and 203-bp fragments for each of the nematode species. Detection times for nematode DNA within the guts of Collembola were longer than in mites, with half-lives (50% of samples testing positive) of 08.75 h and 05.03 h, respectively. F. candida significantly reduced numbers of the nematode H. megidis, with rates of predation of ,0.4 nematode infective juveniles per collembolan per hour over 10 h. Four taxa of field-caught micro-arthropod that had been exposed to the nematode P. hermaphrodita for a period of 12 h were analysed and significant numbers of three taxa tested positive. This is the first application of PCR techniques for the study of nematophagy and the first time these techniques have been used to measure predation on nematodes in the field. [source] Phylogenetic relationships and pathogenicity of Colletotrichum acutatum isolates from grape in subtropical AustraliaPLANT PATHOLOGY, Issue 3 2007M. A. Whitelaw-Weckert The identity of Colletotrichum acutatum as the causal pathogen of grape ripe-rot, which causes yield loss and a bitter taint that lowers wine quality in Australian subtropical wine-grape regions, was confirmed using species-specific primers. Cultural, morphological and molecular methods (RAPD-PCR and sequencing of parts of the 5·8S-ITS regions and the ,-tubulin-2 gene) were used to determine the phylogenetic relationships of Australian C. acutatum isolates from wine grapes and other horticultural crops. A combination of RAPD-PCR and ,-tubulin-2 gene data showed that all wine-grape ripe-rot isolates from northern regions of New South Wales (NSW) and Queensland belong to a proposed new C. acutatum group (A9), together with isolates from Australian strawberry, mango, blueberry and olive. The 5·8S-ITS sequences for these grape pathogens were identical to published sequences for an isolate from Cyclamen (the Netherlands) and differed by 1 bp from isolates from Capsicum (Taiwan) and orange (Costa Rica). The grape ripe-rot isolates from the Shoalhaven Valley (southern NSW) were clustered within two other C. acutatum groups: A2 and A5. In vitro infection studies showed that Australian C. acutatum isolates from almond, blueberry, chilli, grape, mango, olive, strawberry and tomato were able to infect grape and could also infect blueberry and strawberry, indicating a lack of host specificity. This lack of host specificity, the genetic similarity with non-grape isolates, and the fact that many of the non-grape hosts were isolated from wine-growing regions, suggest the potential for cross-infection between grape and other horticultural crops. [source] Development of species-specific primers for the ectoparasitic nematode species Xiphinema brevicolle, X. diffusum, X. elongatum, X. ifacolum and X. longicaudatum (Nematoda: Longidoridae) based on ribosomal DNA sequencesANNALS OF APPLIED BIOLOGY, Issue 3 2005CLAUDIO M G OLIVEIRA Summary The objective of this study was to develop single-step PCR species-specific primers that reliably discriminate four economically important Xiphinema species (X. brevicolle, X. elongatum, X. ifacolum and X. longicaudatum) and X. diffusum that is taxonomically very similar to X. brevicolle. Each species-specific reverse primer was located in the ITS-1 rDNA region and was used in combination with a universal forward primer located in the 18S rDNA gene. Primer reliability was confirmed by screening seven and 11 populations, respectively of X. diffusum and X. elongatum. Potential species-specific primers were also identified for X. brevicolle, X. longicaudatum and X. ifacolum, however too few populations of these species were available to thoroughly assess their reliability. For all species-specific primers, specificity was demonstrated by the absence of cross-reactions with 14 non-target Xiphinema species. Multiplex PCR was effective and reproducible for two (X. longicaudatum and X. ifacolum) or three (X. brevicolle, X. diffusum and X. elongatum) of the target nematode species, thus improving the applicability of the diagnostic primers. [source] Molecular identification of coliform bacteria from colicky breastfed infantsACTA PAEDIATRICA, Issue 10 2009F Savino Abstract Objective:, To determine the presence of intestinal coliform bacteria in colicky vs healthy infants. Study design:, We isolated coliform strains from faeces and performed quantitative bacterial cultures in 41 colicky and 39 healthy breastfed infants, identified using PCR with species-specific primers, strain-specific Automated Ribotyping and the API-50E kit for Enterobacteriaceae to identify the most frequent strains. Results:, Coliform strains were more abundant in colicky infants (median 6.04 log10 CFU/g faeces, range 2.00,8.76) vs controls (median 4.47 log10 CFU/g faeces, range 1.00,8.08) (p = 0.026). Escherichia coli, Klebsiella pneumoniae, K. oxytoca, Enterobacter cloacae, E. aerogenes and Enterococcus faecalis were the predominant species in colicky and healthy infants. The counts of each bacterial species differed between the two groups, and the difference was significant (p = 0.002) for E. coli: median 6.30 log10 CFU/g faeces (range 3.00,8.74) in colicky infants, and median 4.70 log10 CFU/g faeces (range 2.00,5.85) in controls. Conclusions:, This is the first study to evaluate the colonization patterns of gas-forming coliforms in colicky infants and healthy controls identified by molecular methods. Coliform bacteria, particularly Escherichia coli, were found to be more abundant in colicky infants. Our data could help to shed light on the cause of infantile colic. [source] | |||||||||||||||||||||||||