JOURNAL OF FOOD QUALITY, Issue 6 2004
ANGELA DI PINTO
ABSTRACT The authors used a polymerase chain reaction (PCR) assay on buffalo mozzarella, a typical Italian dairy product, from the Apulia markets to evaluate the presence of cow milk and verification of the mozzarella label. The results obtained from 30 mozzarella samples demonstrated the presence of the cow genome in 22/30 samples, highlighting contamination as probable fraudulent adding of cow's milk or use of the same equipments in both working cycles. [source]

A NOVEL MULTIPLEX POLYMERASE CHAIN REACTION FOR SIMULTANEOUS DETECTION OF YERSINIA ENTEROCOLITICA, STAPHYLOCOCCUS AUREUS, AEROMONAS AND SALMONELLA FROM CHICKEN MEAT AND MILK SAMPLES

JOURNAL OF FOOD SAFETY, Issue 2 2010
K. BALAKRISHNA
ABSTRACT Yersinia enterocolitica, Staphylococcus aureus, Aeromonas and Salmonella are among the most important foodborne bacterial pathogens. The majority of human infections caused by all of these organisms are associated with ingestion of undercooked and contaminated meat, dairy products and water where in the secreted bacterial toxins lead to foodborne intoxications. We, here, report a new multiplex polymerase chain reaction (mPCR) assay for the simultaneous detection of these important foodborne bacterial pathogens. The mPCR targeted Ail and virF genes of Y. enterocolitica, nuc and entB genes of S. aureus, aerA and 16S rRNA genes of Aeromonas and invA, an invasion protein A gene of Salmonella. An internal amplification control designed to check the false negative reactions in mPCR was also included. This procedure could detect initial populations of 1,100 cfu/g or /mL within 24 h in experimentally spiked food and water samples. When evaluated on a total of 104 naturally occurring food samples, the mPCR detected two samples to contain S. aureus, one was identified to contain Y. enterocolitica and four samples were identified to contain Salmonella species individually. This was compared with the standard microbiological and biochemical identification procedures. PRACTICAL APPLICATIONS All the microorganisms selected in this study are food and waterborne and contaminate a variety of food items. Pathogenic Y. enterocolitica and Aeromonas species are able to grow and multiply and secrete toxins even at low temperatures. The high throughput and cost-effective multiplex polymerase chain reaction method reported here could be a viable alternative for detection of pathogenic Y. enterocolitica, S. aureus, Aeromonas and Salmonella from food and environmental samples. [source]

Caractérisation par PCR de deux souches d'Erwinia carotovora isolées de la rhizosphère de la pomme de terre dans la région du grand Casablanca au Maroc

EPPO BULLETIN, Issue 1 2007
B. Anajjar
Les infections latentes de la pomme de terre causées par les bactéries du genre Erwinia sont responsables de pertes importantes de cette culture, surtout au cours du transport et du stockage au Maroc. Le sol constitue l'un des substrats potentiels dans ces infections. Deux souches bactériennes ont été isolées de la rhizosphère des plantations de pomme de terre dans la province de Mediouna (wilaya du Grand Casablanca). La méthode de PCR (Polymerase Chain Reaction) utilisant les amorces Y1 et Y2 a permis l'identification de ces souches comme étant Erwinia carotovora. Leur pouvoir pathogène a étéévalué sur des tubercules de pomme de terre de la variété Désirée en comparaison avec une souche d'Erwinia carotovora subsp. carotovora isolée de la pomme de terre dans la même rhizosphère. [source]

Direct genotyping of the poplar leaf rust fungus, Melampsora medusae f. sp. deltoidae, using codominant PCR-SSCP markers

FOREST PATHOLOGY, Issue 4 2005
M. Bourassa
Summary Two anonymous DNA markers that are revealed by single-strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono-uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono-uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re-amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono-uredinial isolates of Mmd tested. From the overall 40 mono-uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse-grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies. Résumé Deux marqueurs génétiques anonymes, révélés par analyse SSCP (Single-Strand Conformational Polymorphism) ont été développés afin de détecter des polymorphismes génétiques chez le Melampsora medusae f. sp. deltoidae (Mmd). Dans un premier temps, des isolats mono-urédiniaux ont été obtenus, puis l'ADN a été extrait à partir des urédiniospores, les produits d'amplification RAPD (Random Amplified Polymorphic DNA) ont été générés à partir de huit de ces isolats mono-urédiniaux et les résultats d'amplification ont par la suite été séparés sur gel SSCP afin d'identifier des polymorphismes entre les isolats. Les bandes sur gel SSCP représentant des loci polymorphiques putatifs entre les isolats ont été prélevées du gel, ré-amplifiées par la technique d'amplification PCR (Polymerase Chain Reaction), clonées, puis séquencées. Pour deux fragments d'ADN séquencés sur un total de trois, une paire d'amorces a été développée afin de permettre l'amplification d'un fragment de taille adéquate pour analyse SSCP (<600 pb). Chaque paire d'amorces a produit un signal d'amplification positif pour chacun des huit isolats à l'origine de ces nouvelles amorces; les produits PCR ont ensuite été analysés par la technique SSCP. Les deux loci putatifs ont révélé des polymorphismes génétiques entre les isolats. Les deux paires d'amorces ont produit un fragment d'amplification de la taille attendue pour chacun des 32 isolats mono-urédiniaux supplémentaires testés. Des 40 isolats testés, 5 et 11 allèles ont été détectés, alors que 12 et 34 isolats se sont révélés hétérozygotes (tel qu'indiqué par la présence de plus de deux bandes sur gel SSCP) pour les loci A et B, respectivement. La spécificité des deux paires d'amorces a été testée à partir de 106 isolats fongiques appartenant à différents groupes taxonomiques, incluant d'autres rouilles, de même qu'à partir de l'ADN extrait de feuilles de peupliers cultivés en serre. Un signal d'amplification positif n'a été obtenu qu'en présence d'ADN du Mmd. Les conditions d'amplification PCR ont été optimisées pour les deux paires d'amorces développées afin de permettre le génotypage directement à partir d'urédinies individuelles prélevées sur des feuilles de peuplier infectées. La possibilité de génotyper directement des urédinies individuelles permet d'éviter l'obligation de cultiver le champignon pour génotyper les individus, ce qui représente un avantage important des marqueurs génétiques développés ici, puisqu'il devient dès lors possible de traiter un grand nombre d'échantillons lors de la réalisation d'études de populations. Zusammenfassung Zum Nachweis von Polymorphismen bei Melampsora medusae f. sp. deltoidae wurden zwei anonyme DNA Marker aus einer SSCP-Analyse entwickelt. Zunächst wurden Isolate aus einzelnen Uredinien gewonnen, die DNA wurde aus den Uredosporen extrahiert und polymorphe RAPD, Amplifikationsprodukte von acht Mono-Uredinium-Isolaten wurden auf einem SSCP-Gel getrennt, um Unterschiede zwischen ihnen nachzuweisen. Banden, die bei den acht geprüften Isolaten mögliche polymorphe Loci darstellten, wurden aus dem SSCP-Gel ausgeschnitten und mit PCR reamplifiziert, dann geklont und sequenziert. Für zwei von insgesamt drei sequenzierten DNA-Fragmenten wurde ein Primerpaar entwickelt, um ein in der Grösse für die SSCP-Analyse (<600 bp) geeignetes DNA-Fragment zu amplifizieren. Jedes Primerpaar amplifizierte bei allen acht ursprünglich für ihre Entwicklung verwendeten Isolaten ein PCR-Produkt, und diese wurden anschliessend mit SSCP analysiert. Für beide putativen Loci wurden bei den Isolaten Polymorphismen festgestellt. Die beiden Primerpaare amplifizierten ein PCR-Produkt der erwarteten Grösse bei allen 32 zusätzlich geprüften Mono-Uredinium-Isolaten des Pilzes. Bei den insgesamt 40 geprüften Mono-Uredinium-Isolaten wurden für die Loci A und B 5 bzw. 11 Allele gefunden, und 12 bzw. 34 Isolate erwiesen sich als heterozygot, was durch mehr als zwei Banden auf den SSCP-Gelen angezeigt wurde. Die Spezifität der Primerpaare wurden mit 106 Pilzisolaten aus verschiedenen Taxa geprüft, darunter andere Roste sowie DNA aus gesunden Pappelblättern aus Gewächshauskulturen. DNA-Amplifikationsprodukte der erwarteten Grösse wurden nur erhalten, wenn DNA von Melampsora medusae f. sp. deltoidae präsent war. Die PCR-Amplifikations-Bedingungen mit diesen beiden Primerpaaren wurde so optimiert, dass ein Genotyping direkt bei einzelnen von infizierten Blättern entnommenen Uredinien erfolgen kann und somit eine Pilzkultur zur Charakterisierung von Individuen entfällt. Dies ermöglicht grosse Probenzahlen in Populationsstudien. [source]

The analysis of JAK2 and MPL mutations and JAK2 single nucleotide polymorphisms in MPN patients by MassARRAY assay

INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2010
S.-J. ZHANG
Summary Recent studies have shown that JAK2 V617F, MPL W515L/K and JAK2 exon 12 mutations underlie the major molecular pathogenesis of myeloproliferative disorders (MPN). Allele-Specific Polymerase Chain Reaction (AS-PCR), direct sequencing and MassARRAY assay were used to ascertain the real prevalence of these mutations and the influence of genetic susceptibility in Chinese MPN patients. The positive rate of JAK2 V617F in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) was 82.0%, 36.6% and 51.1% respectively. One ET patient and two PMF patients harboured the MPL W515L mutation and three PV patients harboured JAK2 exon 12 mutations. All of these patients were confirmed as JAK2 V617F negative. Clinical data demonstrated that PV patients with JAK2 exon 12 mutations were younger, had higher haemoglobin levels and white blood cell counts than PV patients with JAK2 V617F. In addition, through analysis of 4 polymorphic loci of JAK2 gene, no significant difference of distribution frequency was found among PV, ET and PMF patients. Distribution frequency of haplotype also was not significantly different among PV, ET and PMF patients. We conclude that JAK2 V617F is a major molecular pathogenesis in Chinese MPN patients. MPL W515L mutation and JAK2 exon 12 mutations can also be found in JAK2 V617F negative MPN patients. [source]

Chronic administration of valproic acid inhibits PC3 cell growth by suppressing tumor angiogenesis in vivo

INTERNATIONAL JOURNAL OF UROLOGY, Issue 9 2007
Dexuan Gao
Aim: Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. We investigated the mechanisms of chronic valproic acid (VPA) inhibiting PC3 cell growth in the study. Methods: We established tumor xenografts of the PC3 cell line and investigated the effect of VPA chronic administration on tumor growth. Apoptosis in tumor tissue was measured using the TUNEL Detection Kit. We detected the effect of VPA chronic administration on histone acetylation; p21CIP1/WAF1 gene expression; vascular endothelial growth factor (VEGF) expression by reverse-transcription Polymerase Chain Reaction (PCR) analysis; immunohistochemistry; and Western Blotting. Result: In mouse models with established subcutaneous prostate (PC3), VPA treatment induced 70% inhibition of tumor growth without overt toxicity. Our result showed that chronic administration of VPA has an effect on tumor growth arrest and the effect was associated with increased histone acetylation, p21CIP1/WAF1 up-regulation, and VEGF down-regulation. Conclusion: We conclude that chronic VPA results in profound decreases in the proliferation of PC3 cells, not only by increasing histone H3 acetylation and up-regulating p21CIP1/WAF1 expression, but also by down-regulating VEGF. [source]

Molecular detection and , -glucuronidase expression of gus -marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings

JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2003
E. Tsomlexoglou
Abstract Aim: To detect L-form bacteria in developing Chinese cabbage seedlings. Methods and Results: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding , -glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of , -glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. , -Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. Conclusions: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. Significance and Impact of the Study: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association. [source]

Detection by PCR of adhesins genes and slime production in clinical Staphylococcus aureus

JOURNAL OF BASIC MICROBIOLOGY, Issue 4 2008
Tarek Zmantar
Abstract The presence of the ica loci and adhesins genes in clinical Staphylococcus aureus strains were considered important factors of virulence. In this study, 46 strains of Staphylococcus aureus were isolated from auricular infection, and were investigated for slime production using Congo Red Agar method (CRA). In order to detect the adhesins genes (ica A, ica D, fnb A, cna, Clf A) Polymerase Chain Reaction was used. Qualitative biofilm production of S. aureus using CRA plates revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were ica A and ica D positive. While the fnbA gene was present in 76.1% of isolated strains. Furthermore, 56.5% of strains have the cna gene and 30.4% were clfA positives. Overall this study confirms the presence of fnb A and ica A/ica D genes in the majority of studies S. aureus strains isolated from Staphylococcal sepsis. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source]

Expression of RNAs encoding for , and , integrin subunits in periodontitis and in cyclosporin A gingival overgrowth

JOURNAL OF CLINICAL PERIODONTOLOGY, Issue 11 2003
A.-L. Bolcato-Bellemin
Abstract Background: Variation of integrin expression in healthy and diseased gingiva revealed a potential biological role for these cell matrix receptors during gingival remodeling. Aim: Here we determined the level of RNA and tissue localization of different integrin subunits in periodontitis and cyclosporin A-induced gingival overgrowth. Methods: The level of expression was determined by Reverse Transcriptase Polymerase Chain Reaction in 12 periodontitis-affected patients, four patients exhibiting severe cyclosporin A-induced gingival overgrowth and seven healthy patients as controls. Results: The RNA encoding for ,1, ,2 and ,5 integrin subunits were reduced in periodontitis gingiva. The reduction observed was stronger in cyclosporin A-treated patients as compared to the healthy controls, while RNA encoding for ,1 subunit was increased. The RNA encoding for ,6 integrin was only reduced in cyclosporin A-treated gingiva. Immunohistochemistry showed that i) integrin ,2 expression is restricted to the gingival epithelium of cyclosporin A-treated patients, ii) the reduction of ,6 integrin expression in cyclosporin A-treated gingiva is due to loss of expression at focal contacts and iii) ,1 integrin is evenly distributed in the three populations with an intensity decrease in periodontitis and cyclosporin A-treated gingiva. Conclusion: Taken together these results showed a role for the integrin receptors in periodontal diseases and cyclosporin A-induced gingival overgrowth. Zusammenfassung Die Variation der Integrin Expression bei gesunder und erkrankter Gingiva zeigt eine potentielle biologische Rolle für diese Zellmatrixrezeptoren während der gingivalen Erneuerung. Wir bestimmten hier die Level von RNA und die Gewebelokalisation von unterschiedlichen Integrin Untereinheiten bei Parodontitis und Cyclosporin A induzierter gingivaler Wucherung. Die Level der Expression wurden mit der reversen Transscriptase Polymerase Kettenreaktion bei 12 Parodontitis-Patienten, 4 Patienten mit schwerer Cyclosporin A induzierter gingivaler Wucherung und sieben gesunden Kontrollpatienten bestimmt. Die kodierende RNA für ,1, ,2 und ,5 Integrin Untereinheiten waren in der Gingiva mit Parodontitis reduziert. Die beobachtete Reduktion war stärker bei den mit Cyclosporin A behandelten Patienten verglichen mit den gesunden Kontrollen, während kodierende RNA für ,1 Untereinheiten erhöht war. Die kodierende RNA für ,6 Integrin war nur bei der Cyclosporin A behandelten Gingiva reduziert. Die Immunhistochemie zeigte (i) die Integrin ,2 Expression ist auf das gingivale Epithel von Cyclosporin A behandelten Patienten beschränkt, (ii) die Reduktion von ,6 Integrin Expression bei Cyclosporin A behandelter Gingiva ist die Folge von fokalen Expressionverlusten und (iii) ,1 Integrin ist gleichmäßig verteilt in den drei Populationen mit einer Intensitätsabnahme bei Parodontitis und Cyclosporin A behandelter Gingiva. Zusammenfassend zeigen die Ergebnisse eine Rolle für die Integrin Rezeptoren bei den parodontalen Erkrankungen und Cyclosporin A induzierter gingivalen Wucherung. Résumé La variation d'expression des intégrines dans les tissus gingivaux sain et pathologique a démontré le rôle biologique potentiel de ces récepteurs de la matrice extracellulaire au cours du remodelage tissulaire gingival. La quantité d'ARN et la localisation tissulaire de certaines sous-unités d'intégrines dans la parodontite et l'hyperplasie gingivale induite par la ciclosporine A ont été déterminées. Le niveau d'expression a étéévalué par transcription inverse des ARN et réaction de polymérisation en chaine chez douze patients atteints de parodontite, quatre patients présentant une hyperplasie gingivale sévère induite par la ciclosporine A et sept patients sains ayant servi de témoins. L'expression des ARN codant pour les sous-unités ,1, ,2 et ,5était diminuée dans le tissu gingival atteint de parodontite. La diminution observée était plus importante chez les patients traités par la ciclosporine A, comparée aux témoins sains alors que l'expression de l'ARN codant pour la sous-unité,1était augmentée. L'expression de l'ARN codant pour la sous-unité,6était diminuée uniquement dans le tissu gingival traité par la ciclosporine A. L'immohistochimie a montré que (1) l'expression de la sous-unité,2 est limitée à l'épithélium gingival des patients traités par la ciclosporine A, (2) la diminution de l'expression de la sous-unité,6 dans le tissu gingival traité par la ciclosporine A est due à une perte des contacts focaux et (3) la sous-unité,1 est répartie de manière uniforme dans les trois groupes avec une diminution de l'intensité dans les cas de parodontite et d'hyperplasie gingivale induite par la ciclosporine A. Ces résultats montrent un rôle des récepteurs de type intégrine dans la pathologie parodontale et l'hyperplasie gingivale induite par la ciclosporine A. [source]

Evaluation of a Polymerase Chain Reaction,Based System for Detecting Salmonella Species from Pork Carcass Sponge Samples

JOURNAL OF FOOD SCIENCE, Issue 3 2003
Chih-Chuan Wu
ABSTRACT: Pork carcass sponge samples (n = 230) collected from 10 Taiwanese slaughter plants were screened for Salmonella using 2 methods: BAX® for Screening/Salmonella, a polymerase chain reaction-based detection system and a culture method using SM-ID agar as the selective plating medium. The BAX method identified 14 samples as positive for Salmonella. The 8 samples identified as Salmonella-positive by the SM-ID method were also BAX-positive. Inoculation studies showed BAX detected Salmonella in samples having initial 1.4 × 101 cfu/mL Salmonella inoculum prior to the enrichment process in the presence of 3.0 × 106 cfu/mL of non- Salmonella florae. BAX provides a rapid screening alternative to the culture method for Salmonella detection. [source]

Southwest China Han Population Data for Nine Y-STR Loci by Multiplex Polymerase Chain Reaction

JOURNAL OF FORENSIC SCIENCES, Issue 1 2007
Meisen Shi Ph.D.
POPULATION: One hundred and twenty unrelated Han ethnic individuals from Chengdu, southwest China. [source]

Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 6 2009
Yin-Wan Wendy Fung
Abstract Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice. We combined the RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice. We believe that the findings described here will help to elucidate the molecular mechanism(s) underlying the developmental processes of rice leaf. [source]

Newborn screening in Fragile X syndrome

JOURNAL OF INTELLECTUAL DISABILITY RESEARCH, Issue 10 2008
F. Tassone
Background: Screening for the FMR1 mutations has been a topic of considerable discussion since the FMR1 gene was identified. However, Fragile X has not been recommended for newborn screening mainly because of the lack of an accurate screening test and of data on potential benefits. We have recently developed an improved Polymerase Chain Reaction (PCR) method for the identification of premutation and full mutation alleles for the FMR1 gene. Method: The method is inexpensive, accurate and quick and can be performed on a number of sample templates including, importantly, blood spots. We have applied this method for international screening. Specifically, we have screened 5267 anonymous blood spot samples from newborn males from the centre-northwest region of Spain. We have also used this technology to a pilot ,high risk' screening program of individuals with autism and/or intellectual disabilities and family members of a proband with fragile X initiated in Guatemala. This project is a prototype for future screening endeavours. Results: One important outcome from this study is that the frequency of premutation alleles (1 per 250) appears to be higher than previously reported. This is of importance, especially in view of the different phenotypic involvement observed in carriers of premutation alleles, including neurological problems such as FXTAS. Here, we present data on the frequency of premutation/full alleles found in this population and their size distribution. Conclusion: This project is a prototype for future screening endeavours. Results from our pilot program in both Spain and Guatemala will lend strong support for implementing this technology for rapid screening to a much larger scale population screening. [source]

Presence and expression of JCV early gene large T Antigen in the brains of immunocompromised and immunocompetent individuals

JOURNAL OF MEDICAL VIROLOGY, Issue 12 2008
Serena Delbue
Abstract JC virus (JCV) is a polyomavirus that asymptomatically infects up to 80% of the worldwide human population and establishes latency in the kidney. In the case of host immunodeficiency, it can cause progressive multifocal leukoencephalopathy (PML), which is a fatal demyelinating disease of the central nervous system. In an attempt to understand better PML pathogenesis and JCV infection, the presence of the JCV genome and expression of the early viral protein in the brain of deceased individuals, with and without HIV infection, was investigated. Sixty autopsy samples of brain tissues were collected from 15 HIV-positive PML patients, 15 HIV-positive patients with other neurological diseases, 15 HIV-positive patients without neurological disorders, and 15 HIV-negative individuals who died from diseases unrelated to the central nervous system. By means of specific Real Time Polymerase Chain Reaction, the JCV genome was detected in 14 of 15 PML brains, three of 15 HIV-positive brains (with and without neurological diseases), and 1 of 15 HIV-negative brains. JCV genotyping was also performed. Expression of the early JCV protein T Antigen was verified by a specific immunohistochemistry assay, and it was found in the brain tissues from 12 PML cases and one case with other neurological disease. The data obtained demonstrate that infection of the brain with JCV can also be observed in the brains of HIV-negative individuals, without neurological disorders. However, viral protein expression was limited to PML brains and to one brain from a patient with other neurological disease, suggesting that JCV can also be present in the brains of patients without PML. J. Med. Virol. 80:2147,2152, 2008. © 2008 Wiley-Liss, Inc. [source]

Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain Reaction

JOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2009
Xiaojie Wang
Abstract Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst. Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst. In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants. [source]

A Quantitative Polymerase Chain Reaction Assay for the Detection of Polyscytalum pustulans, the Cause of Skin Spot Disease of Potato

JOURNAL OF PHYTOPATHOLOGY, Issue 3 2009
A. K. Lees
Abstract Skin spot disease of potato caused by the pathogen Polyscytalum pustulans is likely to become more important with the withdrawal of 2-aminobutane as a fungicide, and new methods of control will need to be found. As part of a disease control strategy, it will be necessary to study the disease in more detail, to utilize host resistance and to identify stocks where problems are likely to arise. Existing methods for the detection and quantification of P. pustulans are time-consuming and require specific expertise. Real-time PCR assays have been developed for many pathogens of potato and have subsequently been used as tools for the study of the epidemiology and control of disease. The development of a real-time PCR assay for the detection and quantification of P. pustulans is described. The specificity of the assay was demonstrated and detection was shown to be reliable at levels as low as 20,250 fg/,l DNA, (equivalent to 60,680 pg DNA/g) in soil and on symptomless tubers at attogram (ag) levels. These values are in line with previously developed tests. [source]

Detection of Heterobasidion annosum s. l. [(Fr.) Bref.] in Norway Spruce by Polymerase Chain Reaction

JOURNAL OF PHYTOPATHOLOGY, Issue 7 2002
G. Bahnweg
Abstract Internal transcribed spacer (ITS) sequences of the rDNA repeat unit of Heterobasidion annosum were used to design specific primers for the detection and quantification of this important forest pathogen by polymerase chain reaction (PCR). Specificity of detection was cross-checked against a variety of other fungi (saprophytes, root pathogens, mycorrhizal fungi) which may occur in the same environment. As little as 1 pg fungal DNA (equiv. to 10,40 genomes) could be detected in 200 ng spruce root DNA (from 1 mg fresh spruce root). The Heterobasidion -specific primers allowed simultaneous detection of Armillaria spp. in multiplex PCR. The method was successfully applied to increment cores of Norway spruce from the forest region Tharandter Wald (Saxonia, Germany), Oberbärenburg (East Ore Mountains, Saxonia) and Oberschleissheim (north of Munich, Bavaria). [source]

Simultaneous PCR Detection of the Two Major Bacterial Pathogens of Geranium

JOURNAL OF PHYTOPATHOLOGY, Issue 2 2002
D. L. GLICK
Xanthomonas campestris pv. pelargonii (Xcp) and Ralstonia solanacearum (Rs) are the two most important bacterial pathogens of commercially cultivated geraniums (Pelargonium spp.), both causing bacterial wilt and leaf spot. Asymptomatic infections are important reservoirs of infections in commercial growing facilities. Our objective was to design a multiplex PCR (Polymerase Chain Reaction) assay to detect infection by either or both of these pathogens. We used a previously characterized PCR primer pair for Xcp that amplifies a region of 200 bp. In addition, we designed a new primer pair specific for Rs that amplifies a region of 822 bp. With these two primer pairs, we could detect either or both pathogens. As geranium tissue extracts frequently contain inhibitors of the PCR process, a negative PCR could result from either an accurate indication that the plant was pathogen-free or from a false negative assay. We therefore designed `amplification competence' primers, targeting a portion of the geranium 18 s rRNA gene, and generating a 494-bp amplification product that confirms amplification competence and validates a negative assay result. Thus, the triple primer pair multiplex PCR screens for the two most important bacterial pathogens of geraniums simultaneously confirms amplification competence for each geranium sample. [source]

Detection of Ralstonia solanacearum in Potato Tubers by Polymerase Chain Reaction

JOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2000
K.-H. Pastrik
Abstract A new polymerase chain reaction (PCR) assay was developed for the detection of Ralstonia solanacearum in potato tubers. The designed primers PS-1/PS-2 based on the sequence data of the 16S rRNA gene. Using the optimized PCR protocol, it was possible to detect R. solanacearum cells artificially added to concentrated potato extracts in the range of 1,10 colony-forming units (CFU) per PCR reaction mixture (10,100 CFU/ml potato homogenate). No amplification products were obtained, when bacteria belonging to other species or genera were submitted to PCR under the same conditions. A total of 10 different DNA extraction methods were adapted for the isolation of R. solanacearum DNA from potato homogenates and were compared for their suitability as pre-PCR procedures. Zusammenfassung Es wurde ein neuer PCR-Test entwickelt für die Detektion von Ralstonia solanacearum in Kartoffel-Knollen. Die entwickelten Primer PS-1/PS-2 basierten auf Sequenzdaten des 16S rRNA Gens. Mit dem optimierten PCR Protokoll war es möglich künstlich zugegebene R. solanacearum Zellen in konzentrierten Kartoffel-Homogenaten zu detektieren, bei einer Nachweis-Empfindlichkeit von 1,10 CFU pro PCR-Mix (10,100 CFU pro ml Kartoffel-Homogenat). Mit dem optimierten PCR Protokoll wurden keine Amplifikationsprodukte bei Bakterien anderer Arten oder Gattungen erhalten. Außerdem wurden 10 unterschiedliche DNA-Extraktionsmethoden getestet zur Isolierung von Ralstonia solanacearum DNA aus Kartoffel-Homogenat und ihre Eignung für die PCR verglichen. [source]

Real-Time Polymerase Chain Reaction: A Novel Molecular Diagnostic Tool for Equine Infectious Diseases

JOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 1 2006
N. Pusterla
The focus of rapid diagnosis of infectious disease of horses in the last decade has shifted from the conventional laboratory techniques of antigen detection, microscopy, and culture to molecular diagnosis of infectious agents. Equine practitioners must be able to interpret the use, limitations, and results of molecular diagnostic techniques, as they are increasingly integrated into routine microbiology laboratory protocols. Polymerase chain reaction (PCR) is the best-known and most successfully implemented diagnostic molecular technology to date. It can detect slow-growing, difficult-to-cultivate, or uncultivatable microorganisms and can be used in situations in which clinical microbiology diagnostic procedures are inadequate, time-consuming, difficult, expensive, or hazardous to laboratory staff. Inherent technical limitations of PCR are present, but they are reduced in laboratories that use standardized protocols, conduct rigid validation protocols, and adhere to appropriate quality-control procedures. Improvements in PCR, especially probe-based real-time PCR, have broadened its diagnostic capabilities in clinical infectious diseases to complement and even surpass traditional methods in some situations. Furthermore, real-time PCR is capable of quantitation, allowing discrimination of clinically relevant infections characterized by pathogen replication and high pathogen loads from chronic latent infections. Automation of all components of PCR is now possible, which will decrease the risk of generating false-positive results due to contamination. The novel real-time PCR strategy and clinical applications in equine infectious diseases will be the subject of this review. [source]

Evaluation of a p30 Gene-Based Real-Time Reverse Transcriptase Polymerase Chain Reaction Assay for Detection of Feline Caliciviruses

JOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 1 2004
Brian A. Scansen
This report describes a feline calicivirus (FCV) p30 gene-based real-time SYBR Green I reverse transcriptase polymerase chain reaction (RT-PCR) assay that is capable of detecting low virus concentrations and a broad range of FCV isolates. The assay consisted of a 1-step RT-PCR reaction with primers delineating a 126-base-pair (bp) region of the FCV p30 gene. Sensitivity of the RT-PCR assay was determined to be equivalent to a FCV titer of 1.2 × 101 to 1.2 × 102 TCID50/mL. The assay was linear over a wide range of template concentrations and had a reaction efficiency of 95%. Specific FCV amplification products were detected from 51 wild-type FCV isolates, whereas specific products were not detected from a canine calicivirus, a rabbit calicivirus, and a bovine calicivirus. The primers used in this study amplified a large number of North American FCV isolates and further confirm the diagnostic utility of p30 gene-based real-time RT-PCR for detection of FCV. [source]

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

LETTERS IN APPLIED MICROBIOLOGY, Issue 4 2002
R.S. Peixoto
Aim: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. Methods: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. Results: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. Conclusions, Significance and Impact of the Study: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE. [source]

Validation of Phage T7 Biological Dosimeter by Quantitative Polymerase Chain Reaction Using Short and Long Segments of Phage T7 DNA ,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2003
M. Hegedüs
ABSTRACT Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts. [source]

Rapid prenatal diagnosis of aneuploidies and zygosity in multiple pregnancies by amniocentesis with single insertion of the needle and quantitative fluorescent PCR

PRENATAL DIAGNOSIS, Issue 8 2003
Vincenzo Cirigliano
Abstract Objective To investigate amniotic fluid (AF) samples retrieved in multiple pregnancies by single insertion of the needle, for rapid assessment of chromosome copy number, zygosity, and cross-contamination between fetuses, using Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) amplification of highly polymorphic microsatellite markers. Methods Fifty-two multiple pregnancies were selected (47 twins, 5 triplets) and 108 samples of amniotic fluid were sampled between 12 to 20 weeks of gestation (mean 15.5) using the single-needle technique. Aneuploidy screening by QF-PCR amplification of short tandem repeats (STRs) on chromosomes X, Y, 21, 13, and 18 was carried out within 24 h of collection. Owing to the sampling procedure, the eventual presence of contamination between fetuses was also evaluated in every case. Results Normal and aneuploid fetuses were readily identified by QF-PCR. Fetal reduction was made available, for trisomic fetuses, without further waiting for completion of fetal karyotyping. In twin gestations, the ultrasound examination of chorionicity was always in agreement with the molecular assessment of zygosity. Contamination between fetuses due to the sampling procedure with a single puncture was never observed. Conclusion Rapid prenatal diagnosis of aneuploidies by QF-PCR is a sensitive, efficient, and reliable assay. When applied in multiple pregnancies, it has the added value of allowing the assessment of zygosity in all cases, independently of chorionicity and fetal sex. Copyright © 2003 John Wiley & Sons, Ltd. [source]

A Survey of Polymerase Chain Reaction (PCR) Amplification Studies of Unicellular Protists Using Single-Cell PCR

THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 5 2009
DENIS H. LYNN
ABSTRACT. We surveyed a variety of studies that have used single-cell polymerase chain reaction (SC-PCR) to examine the gene sequences of a diversity of unicellular protists. Representatives of all the Super-Groups of eukaryotes have been subjected to SC-PCR with ciliates and dinoflagellates being most commonly examined. The SC-PCR was carried out either by directly amplifying a single lysed cell or by first extracting DNA and following this with amplification of the DNA extract. Cell lysis methods included heating, freezing, mechanical rupture, and enzyme digestion. Cells fixed or preserved with ethanol, methanol, and Lugol's have also been used successfully. Heminested or seminested PCR might follow the initial PCR, whose products were then directly sequenced or cloned and then sequenced. The methods are not complicated. This should encourage protistologists to use SC-PCR in the description of new or revised taxa, especially rare and unculturable forms, and it should also enable the probing of gene expression in relation to life history stages. [source]

Congenital Cytomegalovirus Infection Diagnosed by Polymerase Chain Reaction With the Use of Preserved Umbilical Cord in Sensorineural Hearing Loss Children,

THE LARYNGOSCOPE, Issue 11 2006
Hiroshi Ogawa MD
Abstract Objectives/Hypothesis: Congenital cytomegalovirus (CMV) infection is estimated to account for 30% of sensorineural hearing loss (SNHL) cases. Differences in clinical characteristics between CMV-related and unrelated SNHL cases were scrutinized. Methods: Using dried umbilical cord, we have recently developed a polymerase chain reaction (PCR)-based assay for the retrospective detection of congenital CMV infection. Medical records of 7 CMV-related patients identified from 31 SNHL patients by the assay were evaluated for the following: type and degree of hearing impairment, computed tomographic scan results, mental retardation, cerebral palsy, autism, and other multiple disorders. Results: Clinical characteristics of the seven CMV-related SNHL cases were as follows: 1) six of the seven exhibited severe bilateral SNHL, whereas one had severe unilateral SNHL in the right ear. Although the hearing levels of CMV-related patients were more greatly impaired than those of CMV-negative patients, there was no hearing impairment pattern specific to the CMV-related patients; 2) five patients had mental retardation, which was more frequent than in CMV-negative patients; 3) birth weights of the CMV-positive cases were relatively lower. Discussion: Although CMV-positive cases are clinically indistinguishable from CMV-negative cases, our PCR system allowed the retrospective diagnosis of CMV-related SNHL. Conclusion: CMV-related SNHL tends to accompany mental retardation and low birth weight more frequently than does CMV-negative SNHL. [source]

Immunohistochemistry and Reverse Transcriptase,Polymerase Chain Reaction as Methods for Diagnostic Determination of Usher Syndrome Type IIa,

THE LARYNGOSCOPE, Issue 7 2004
Edward Cohn
Abstract Objectives/Hypothesis: Patients having null mutations in the USH2A gene do not produce usherin and therefore are not positive for immunohistochemical staining of the usherin protein. Thus, immunostaining for usherin can serve as a reliable diagnostic tool for Usher syndrome type IIa. Study Design: Prospective. Methods: Immunohistochemical staining for usherin was carried out in basement membrane of minor salivary gland tissue from subjects with confirmed Usher syndrome type IIa and from archival minor salivary gland tissue from patients without Usher syndrome as control samples. Quantitative usherin messenger RNA analysis was performed using minor salivary gland biopsy tissue. Results: Five subjects with Usher syndrome type IIa had no immunostaining in minor salivary gland tissue, whereas control minor salivary gland tissue did stain with usherin antibody. No usherin RNA was detected in biopsy specimens from patients with confirmed Usher syndrome IIa. Conclusion: The feasibility was confirmed of diagnosing Usher syndrome type IIa using purified usherin antibody in subjects having two null USH2A mutations. [source]

Epstein-Barr Virus in Head and Neck Cancer Assessed by Quantitative Polymerase Chain Reaction

THE LARYNGOSCOPE, Issue 6 2004
David Goldenberg MD
Abstract Objectives/Hypothesis: Epstein-Barr virus (EBV) has classically been associated with nasopharyngeal carcinoma and Burkitt's lymphoma. Recently, multiple studies have been published linking EBV with oral squamous cell carcinoma and, to a lesser extent, hypopharyngeal and laryngeal tumors. Using a sensitive method of detection, the authors sought to analyze the presence and quantity of EBV DNA in a large cohort of head and neck cancers. Study Design: Retrospective cohort study. Methods: Three hundred head and neck cancer samples exclusive of nasopharyngeal carcinoma were examined for the presence of EBV using quantitative polymerase chain reaction. Eighty-four tumor samples from the larynx, 30 from the hypopharynx, 73 from the oropharynx, and 113 from the oral cavity were analyzed for EBV quantity, which was expressed as the number of viral copies per cell genome. Representative samples, which contained the highest EBV DNA levels, were examined using in situ hybridization. Results were correlated with tumor grade and site and tobacco and alcohol exposure. Results: Three of 300 (1%) tumor samples were overtly positive for EBV DNA (defined as >0.1 copies of viral DNA/cell genome). Five of 300 (2%) tumor samples showed low levels (defined as >0.01 and <0.1 copies of viral DNA/cell genome), and 68 of 300 tumor samples (23%) showed trace levels (defined as < 0.01 copies of viral DNA/cell genome) of EBV DNA. No correlation was found between EBV positivity and tobacco exposure, alcohol exposure, or tumor grade. Conclusion: In the overwhelming majority of head and neck cancers in this North American cohort, EBV did not appear to contribute to growth of a dominant clonal population with integrated EBV genome and was unlikely to be a genetic etiological agent in tumor development. The low quantities of EBV detected in a minority of head and neck cancers may be related to the presence of EBV genome in rare lymphoid or epithelial cells adjacent to the primary head and neck cancer. [source]

Expanded Genetic Alphabets in the Polymerase Chain Reaction,

ANGEWANDTE CHEMIE, Issue 1 2010
Zunyi Yang Dr.
Gesäuberte PCR: DNA-Polymerasen wurden entdeckt, die zwei zusätzliche Nucleotid-,Buchstaben" (Z und P) in ein erweitertes DNA-Alphabet einfügen, sodass Polymerasekettenreaktionen (PCR) mit sechs Buchstaben möglich werden. Wenn der Einbau in externe Primer bei einer dreifachen Multiplex-PCR betrachtet wurde, lieferten Primer, die Z und P enthielten, deutlich sauberere Resultate. [source]

Real-time Polymerase Chain Reaction to Follow the Response of Muscle to Training

ARTIFICIAL ORGANS, Issue 8 2008
Lauren M. Moore
Abstract:, The adaptive response of muscle to changes in activity or loading can take many weeks. Changes in the levels of RNA within a muscle fiber can give an early indication of the nature of the response of that fiber to changes in activity or loading. We have designed a new primer set for quantitative polymerase chain reaction (PCR) that will allow us to follow these early transcriptional changes in rat muscle, and have shown that analysis can be performed by standard techniques on as little as 5 mg of muscle, an amount that can be obtained by needle biopsy. [source]