Can Application of Universal Primers Alone Be a Substitute for Airborne-Particle Abrasion to Improve Adhesion of Resin Cement to Zirconia?

Purpose: To determine whether universal primers alone can deliver similar levels of adhesion of resin cement to zirconia ceramic when compared to their application in conjunction with airborne-particle abrasion.Materials and Methods: Sintered zirconia blocks (N = 160) (Lava, 3M ESPE), (5.25 x 5.25 x 3 mm(3)) were embedded in acrylic resin, polished, and randomly distributed into 16 groups (n = 10 per group), according to the factors "universal primer" (8 levels) and "air-particle abrasion" (2 levels): 1. ctr: control, without application of a universal primer; 2. AP: Alloy Primer; 3. MP: Monobond Plus; 4. MZP: Metal Zirconia Primer; 5. MZ: MZ Primer; 6. Sg: Signum Zirconia Bond; 7. SbU: Singlebond Universal; 8. ZP: Z Prime Plus. The universal primers were also used after air abrasion (A) of zirconia to form the following 8 groups: Ctr-A, AP-A, MP-A, MZP-A, MZ-A, Sg-A, SbU-A, and ZP-A. After ultrasonic cleaning, air abrasion was performed using Al2O3 particles (110 mu m, 2.5 bar, 20 s at 10 mm) in a chairside air-abrasion device. After ultrasonic cleaning again, universal primers were applied according to each manufacturer's recommendation. The resin cement (RelyX ARC, 3M ESPE) was built up incrementally and photo-polymerized on the zirconia surface using a silicone mold (empty set = 3.5, height = 3 mm). All specimens were stored in distilled water (60 days at 37 degrees C) and then subjected to shear bond strength testing (SBS) in a universal testing machine (1 mm/min). On a separate set of zirconia specimens, contact angle measurements were made using the sessile drop technique with a goniometer after the application of universal primers on control and air-abraded zirconia surfaces. Data (MPa) were analyzed using one-way ANOVA, Tukey's test, and Student's t-test (alpha = 0.05).Results: When universal primers were used alone, SbU presented significantly higher mean SBS (19.5 +/- 5.8) that did the other primers (0 to 9.9 +/- 6.6) (p = 0.001). When air abraded, the groups AP-A (14.1 +/- 6.1), MP-A (15.9 +/- 5.4), ZP-A (16.9 +/- 7.3), SG-A (19.1 +/- 2.1), SbU-A (12 +/- 1.5) showed significant differences (p = 0.03). Adhesive performance of all universal primers was enhanced after air abrasion, with the exception of the SbU and MZ primers. After air abrasion, contact angle measurements were lower for the each primer (without air abrasion: 28.9 to 83.9; with air abrasion: 27.1 to 63.0), except for MZP.Conclusion: Air abrasion with 110 mu m Al2O3 followed by universal primer application increased the bond strength of tested resin cement to zirconia, with the exception of SbU and MZ.

Microeukaryoticdiversity in the extreme environments of the Iberian PyriteBelt: a comparison between universal and fungi-specific primer sets, temperature gradient gel electrophoresis and cloning

FEMS Microbiology Ecology, Vol. 57, nº 1

A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies

We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae.

Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers

Lettuce mottle virus (LeMoV) and dandelion yellow mosaic virus (DaYMV) infect lettuce in South America and Europe, respectively. LeMoV and DaYMV possess isometric particles, occur at low concentrations in plants and have narrow host ranges. Partial genome sequences of both viruses were obtained using purified viral preparations and universal primers for members of the family Sequiviridae. DaYMV and LeMoV sequences were analyzed and showed identity with other members of the family. Universal primers that detect both viruses and specific primers for LeMoV and DaYMV were designed and used in RT-PCR-based diagnostic assays. These results provide the first molecular data on the LeMoV and DaYMV genomes and suggest that LeMoV is a member of the genus Sequivirus, probably distinct from DaYMV.

Phytoplasma closely related to the pigeon pea witches`-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of Sao Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of Sao Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fDI/rPI was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the Pigeon pea witches`-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these result.,;. With two primers D7f2/D7r2 designed based oil the 16S rDNA Sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and `Candidatus Liberibacter asiaticus`. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results Show that a phytoplasma of group IX is associated with citrus HLB symptoms ill northern, central, and Southern SPs. This phytoplasma has very probably been transmitted to citrus from an external Source of inoculum, but the Putative insect vector is not yet known.

Characterization of dengue virus serotype 1 in epidemics in Porto Velho, Rondônia, in 2001-2003

The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universal primers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98% similarity to sequences from Southeast Asia that were obtained from GenBank.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Genomic characterization of a Brazilian TT Virus isolate closely related to SEN virus-F

SEN virus (SENV) is a circular, single stranded DNA virus that has been first characterized in the serum of a human immunodeficiency virus type 1 (HIV-1)-infected patient. Eight genotypes of SENV (A-H) have been identified and further recognized as variants of TT virus (TTV) in the family Circoviridae. Here we describe the first genomic characterization of a SENV isolate (5-A) from South America. Using 'universal' primers, able to amplify most, if not all, TTV/SENV genotypes, a segment of > 3 kb was amplified by polymerase chain reaction from the serum of an HIV-1 infected patient. The amplicon was cloned and a 3087-nucleotide sequence was determined, that showed a high (85%) homology with the sequence of the Italian isolate SENV-F. Proteins encoded by open reading frames (ORFs) 1 to 4 consisted of 758, 129, 276, and 267 amino acids, respectively. By phylogenetic analysis, isolate 5-A was classified into TTV genotype 19 (phylogenetic group 3), together with SENV-F and TTV isolate SAa-38.

Direct identification of recombinant vaccinia virus plaques by PCR.

A fast method for the identification of recombinant vaccinia viruses directly from individual plaques is described. Plaques are picked, resuspended in PBS-A and processed for PCR using two 'universal' primers. The amplified sequences are analyzed by agarose gel electrophoresis. This procedure allows discrimination between spontaneously arising TK-negative mutants, which do not carry the inserted gene, and the desired TK-negative recombinants resulting from insertional inactivation of the TK gene.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.

Prevalence of Lettuce mosaic virus - common strain on three lettuce producing areas from São Paulo State

LMV is one of the most important pathogens of lettuce worldwide. Based on their ability to overcome the resistance genes mo1¹ and mo1² in lettuce, isolates can be divided in two types: LMV-Most, which can infect and are seed-borne in cultivars containing the mo1 gene and LMV-Common, which do not cause symptoms on these cultivars and are seed transmitted only in susceptible cultivars. To evaluate the occurrence of these two types of LMV isolates, a survey was carried out during 2002-2005 in three lettuce production areas from São Paulo State. Total RNA was used for the diagnosis of LMV isolates by RT-PCR using universal primers for the variable N-terminus of the capsid protein, in the 3' end of the genome. Positives samples were analyzed by a second RT-PCR using specifics primers for LMV-Most isolates designed to amplify a fragment from the central region (CI-VPg) of the genome. A total of 1362 samples showing mosaic symptoms were collected and 504 (37.29 %) were positives for LMV. On susceptible lettuce cultivars, LMV-Common was prevalent (77.3%). LMV-Most was found frequently associated with tolerant (mo1¹) lettuce cultivars. Susceptible cultivars correspond today for most of the area of lettuce production. So, despite the ability of LMV-Most isolates to overcome the resistance provided by the recessive mo1¹ gene, they are not prevalent in the conditions of São Paulo State.

Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges

Conidiobolomycosis is a granulomatous disease caused by the fungus Conidiobolus spp. in humans and animals. Traditional technique for diagnosis of the disease is isolation of the agent associated with the presence of typical clinical signs and pathological conditions. The aim of this study was to describe the development of a specific polymerase chain reaction (PCR) test for Conidiobolus lamprauges to detect the fungus in clinical samples. Samples from suspected animals were collected and submitted to isolation, histopathological analysis and amplification by PCR. DNA from tissues was subjected to PCR with fungi universal primers 18S rDNA gene, and specific primers were designed based on the same gene in C. lamprauges that generated products of about 540 bp and 222 bp respectively. The culture was positive in 26.6% of clinical samples. The PCR technique for C. lamprauges showed amplification of DNA from fresh tissues (80%) and paraffin sections (44.4%). In conclusion, the PCR technique described here demonstrated a high sensitivity and specificity for detection of fungal DNA in tissue samples, providing a tool for the rapid diagnosis of C. lamprauges.

A simple RT-PCR-based strategy for screening connexin identity

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Characterization and applications of two protease enzymes obtained by culture dependent and independent approaches from mangrove sediments

Soil community genomics or metagenomics is employed in this study to analyze the evolutionary related - ness of mangrove microbial community. The metagenomic DNA was isolated from mangrove sediment and 16SrDNA was amplified using universal primers. The amplicons were ligated into pTZ57R/T cloning vector and transformed onto E. coli JM109 host cells. The recombinant plasmids were isolated from positive clones and the insert was confirmed by its reamplification. The amplicons were subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) using three different tetra cutter restriction enzymes namely Sau3A1, Hha1 and HpaII. The 16SrDNA insert were sequenced and their identity was determined. The sequences were submitted to NCBI database and accession numbers obtained. The phylo - genetic tree was constructed based on Neighbor-Joining technique. Clones belonged to two major phyla of the bacterial domain, namely Firmicutes and Proteobacteria, with members of Firmicutes predominating. The microbial diversity of the mangrove sediment was explored in this manner.

In vitro effects of synbiotic fermentation on the canine faecal microbiota.

Stirred, pH-controlled anaerobic batch cultures were used to investigate the in vitro effects of galacto-oligosaccharides (GOS) alone or combined with the probiotic Bifidobacterium bifidum 02 450B on the canine faecal microbiota of three different donors. GOS supported the growth of B. bifidum 02 450B throughout the fermentation. Quantitative analysis of bacterial populations by FISH revealed significant increases in Bifidobacterium spp. counts (Bif164) and a concomitant decrease in Clostridium histolyticum counts (Chis150) in the synbiotic-containing vessels compared with the controls and GOS vessels. Vessels containing probiotic alone displayed a transient increase in Bifidobacterium spp. and a transient decrease in Bacteroides spp. Denaturing gradient gel electrophoresis analysis showed that GOS elicited similar alterations in the microbial profiles of the three in vitro runs. However, the synbiotic did not alter the microbial diversity of the three runs to the same extent as GOS alone. Nested PCR using universal primers, followed by bifidobacterial-specific primers illustrated low bifidobacterial diversity in dogs, which did not change drastically during the in vitro fermentation. This study illustrates that the canine faecal microbiota can be modulated in vitro by GOS supplementation and that GOS can sustain the growth of B. bifidum 02 450B in a synbiotic combination.