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Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

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) hybridization probes, previously described by Orosz et al. [ 10 ]. Each experiment included one reaction mixture without DNA as a negative control; positive control and each specimen were run in duplicate for real-time polymerase chain reaction (PCR) assay in

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Acta Microbiologica et Immunologica Hungarica
Authors: Erika Orosz, Nóra Szentmáry, Huba J. Kiss, Ágnes Farkas, István Kucsera, and Zoltán Zsolt Nagy

amplification was performed using genus-specific primers and genus-specific-fluorescence resonance energy transfer hybridization probes, previously described by Orosz et al. [ 14 ]. Each experiment included one reaction mixture without DNA as a negative control

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The multicolour genomic in situ hybridization (mcGISH) method was improved in order to visualize the U b and M b genomes of Aegilops biuncialis Vis. (2n=4x=28, U b U b M b M b ). Hybridization probes prepared from the diploid U and M genome donors, Ae. umbellulata and Ae. comosa , resulted in clear hybridization signals on the U and M chromosomes in Ae. biuncialis . The random primed labelling method made it possible to decrease the blocking ratio to 1:30. McGISH allowed the simultaneous discrimination of individual Ae. biuncialis genomes and wheat chromosomes in γ-irradiated Triticum aestivum-Ae. biuncialis amphiploids (2n=70; AABBDDU b U b M b M b ). Dicentric chromosomes, terminal and interstitial translocations and centric fusions were detected in the irradiated generation. The irradiation-induced wheat- Ae. biuncialis intergenomic translocations will facilitate the successful introgression of useful agronomic traits into bread wheat.

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The DNA of the prototype strains of ovine adenovirus (OAdV) 1 through 5 was analysed by restriction enzyme (RE) digestion. The RE patterns generated by Hindlll and PstI enzymes were characteristic of the examined strains. OAdV-2 and 3 resembled each other the most, and their EcoRI and Hindlll patterns seemed to be identical. Considering the number of comigrating fragments, serotypes OAdV-2, 3, 4 and 5 looked more closely related to each other than to OAdV-1. This finding was strengthened by Southern blot hybridisations probed with random Hindlll clones of OAdV-3. The estimated genome size of the examined OAdV types ranged between 31.9 and 32.8 kilobase pairs. The results supported the new genus classification of OAdVs.

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Acta Microbiologica et Immunologica Hungarica
Authors: Erika Orosz, Ágnes Farkas, László Ködöböcz, Péter Becságh, József Danka, István Kucsera, and György Füleky

Acanthamoeba species are free-living amoebae that can be found in almost every range of environments. Within this genus, a number of species are recognized as human pathogens, potentially causing Acanthamoeba keratitis, granulomatous amoebic encephalitis, and chronic granulomatous lesions. Soil and water samples were taken from experimental station at Julianna Major of Plant Protection Institute of Centre for Agricultural Research, Hungarian Academy of Sciences (CAR HAS). We detected living Acanthamoeba spp. based on culture-confirmed detection combined with the molecular taxonomic identification method. Living Acanthamoeba spp. were detected in thirteen (65%) samples. The presence of Acanthamoeba spp. in the samples depends significantly on the rhizosphere plants. The most frequently identified living Acanthamoeba genotype was T4 followed by T11, T2/T6 and T17. Genotypes T4 and T11 of Acanthamoeba, are responsible for Acanthamoeba keratitis as well as granulomatous amoebic encephalitis, and should therefore be considered as a potential health risk associated with human activities in the environment.

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European Journal of Microbiology and Immunology
Authors: Trudy M. Wassenaar, Anke Zschüttig, Claudia Beimfohr, Thomas Geske, Christian Auerbach, Helen Cook, Kurt Zimmermann, and Florian Gunzer

The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli.

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Orvosi Hetilap
Authors: Bálint Nagy, Levente Lázár, Gyula Richárd Nagy, Zoltán Bán, and Zoltán Papp

transplantation using fluorescence resonance energy transfer hybridization probes. J. Clin. Microbiol., 2000, 38 , 2929–2932. Ernault P. Real-time PCR for diagnosis and follow-up of

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Genomikus kópiaszám-eltérések szűrése krónikus limfoid leukémiában multiplex ligációfüggő szondaamplifikációval

Screening for genomic copy number alterations in chronic lymphocytic leukemia using multiplex ligation-dependent probe amplification

Hematológia–Transzfuziológia
Authors: Richárd Kiss, Gergő Papp, Szilvia Krizsán, Lili Kotmayer, Ambrus Gángó, Noémi Nagy, Bence Bátai, Zoltán Mátrai, Csaba Bödör, and Donát Alpár

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A krónikus limfocitás leukémia klinikailag heterogén megjelenése genetikai diverzitással párosul. Hazánkban fluoreszcens in situ hibridizáció terjedt el a betegekben előforduló, prognosztikai jelentőséggel bíró kiegyensúlyozatlan abnormalitások analíziséhez. A jelenleg szűrt markerek alacsony száma miatt a módszer alábecsülheti a vizsgált leukémiás sejtpopuláció genetikai komplexitását. Tanulmányunkban a multiplex ligációfüggő szondaamplifikáció hatékonyságát vizsgáltuk genomikus kópiaszám-eltérésekkel járó aberrációk kimutatásához krónikus limfocitás leukémiában. Tizennyolc olyan beteg diagnóziskori mintáját analizáltuk SALSA P037 és P038-as szondakeverékekkel, akikben a korábbi diagnosztikai vizsgálat molekuláris citogenetikai eltérést jelzett. Öt, ibrutinibterápiában részesülő betegnél kezelés előtt és azt követően vett mintapárokat vizsgáltunk. Az eredmények egy részét fluoreszcens in situ hibridizációval validáltuk. A két alkalmazott módszerrel összesen 63 aberrációt azonosítottunk 18, célzott terápiában nem részesülő beteg mintájában. A multiplex ligációfüggő szondaamplifikáció 59 eltérést tárt fel, melyből minden olyan aberrációt sikerrel validáltunk (n = 38), melyhez rendelkezésre állt fluoreszcens in situ hibridizációs szonda. Utóbbi módszerrel további 4 eltérés mutatkozott, melyek mintában való reprezentáltsága 20% alattinak bizonyult, ezért nem igazolódott MLPA-val. Az öt, ibrutinibterápiában részesülő beteg közül két betegben jelent meg új kópiaszám-eltérés a kezelés megkezdése utáni mintában, egy esetben a kezelés előtti mintában azonosítható aberrációk eliminálódtak a kezelés során, két esetben pedig egyáltalán nem mutatkozott abnormalitás a mintapárokban. Intézetünkben beállítottuk a multiplex ligációfüggő szondaamplifikációt krónikus limfocitás leukémia vizsgálatához. A módszer hasznos eszköze lehet ezen entitás diagnosztikájának és kutatásának. A jelenleg használt technikákat kiegészítve teljesebb képet adhat a betegség genetikai komplexitásáról és segítheti az egyes betegek pontosabb prognosztikai besorolását.

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References 1. Zengh D , Alm EW , Stahl DA , Raskin L : Characterization of universal small-subunit rRNA hybridization probes for

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